Regeneration of endogenous myocardial tissue

ABSTRACT

This invention provides a method of treating a disorder of a subject&#39;s heart involving loss of cardiomyocytes which comprises administering to the subject a composition comprising an amount of a human stromal derived factor-1 and an amount of a human granulocyte-colony stimulating factor, the composition being administered in an amount effective to cause proliferation of cardiomyocytes within the subject&#39;s heart so as to thereby treat the disorder. This invention also provides a method of treating a subject suffering from a disorder of a tissue involving loss and/or apoptosis of cells of the tissue which comprises administering to the subject a composition comprising an amount of an agent which induces phosphorylation and/or activation of protein kinase B, or an agent which induces phosphorylation and/or activation of an extracellular signal-regulated protein kinase, or an agent which induces activation of CXCR4.

Throughout this application, various publications are referenced inparentheses by arabic numbers. Full citations for these references maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND

Healing of a myocardial infarct is complicated by the need for viablemyocytes at the peri-infarct rim to undergo compensatory hypertrophy inorder to increase pump function in response to the loss of infarctedtissue (1,2). This initiates a process termed cardiac remodelling whichis characterized by apoptotic loss of hypertrophied myocytes, expansionof the initial infarct area, progressive collagen replacement, and heartfailure (3-6). We have recently put forward the hypothesis thathypertrophied cardiac myocytes undergo apoptosis because the endogenouscapillary network cannot provide the compensatory increase in perfusionrequired for cell survival (7).

Vascular network formation is the end result of a complex process thatbegins in the pre-natal period with induction of vasculogenesis byhemangioblasts—cells derived from the human ventral aorta which giverise to both endothelial and hematopoietic elements (8-11). Cells whichcan differentiate into endothelial elements also exist in adult bonemarrow (12-14) and can induce vasculogenesis in ischemic tissues(15-17). In the adult, new blood vessel formation can occur eitherthrough angiogenesis from pre-existing mature endothelium orvasculogenesis mediated by bone marrow-derived endothelial precursors.Recently, we identified a specific population of endothelial progenitorcells (angioblasts) derived from human adult bone marrow which hasphenotypic and functional characteristics of embryonic angioblasts (7).We showed that intravenous administration of these cells resulted inselective homing to ischemic myocardium, induction of infarct bedvasculogenesis, prevention of peri-infarct myocyte apoptosis, andsignificant improvement in myocardial function (7).

We recently discovered that CXC chemokines containing the ELR motifregulate migration of human bone marrow-derived endothelial progenitorcells to sites of tissue ischemia. Moreover, since selective bone marrowhoming and engraftment of hematopoietic progenitors depends on CXCR4binding to SDF-1 expressed constitutively in the bone marrow (28-30), wedemonstrated that interruption of CXCR4/SDF-1 interactions couldredirect trafficking of human bone marrow-derived endothelial progenitorcells to sites of tissue ischemia, thereby augmenting therapeuticvasculogenesis. Our results indicated that CXC chemokines, includingIL-8, Gro-alpha, and SDF-1, play a central role in regulating humanadult bone marrow-dependent vasculogenesis.

Recent observations have suggested that a second compensatory responseof viable cardiomyocytes is to proliferate and regenerate followinginjury (18,19). We have previously shown that pro-angiogenic factors,such as endothelial progenitor cells at a minimum concentration caninduce vasculogenesis. Here we disclose the result that careful dosingof pro-angiogenic agents, or agents that can activate AKT or ERK, oractivate CXCR4 on cells can induce cardiomyocyte proliferation orprevent loss of cardiomyoctyes also.

SUMMARY

This invention provides a method of treating a disorder of a subject'sheart involving loss of cardiomyocytes which comprises administering tothe subject a composition comprising an amount of a human stromalderived factor-1 and an amount of a human granulocyte-colony stimulatingfactor, the composition being administered in an amount effective tocause proliferation of cardiomyocytes within the subject's heart so asto thereby treat the disorder.

This invention also provides a method of treating a subject sufferingfrom a disorder of a tissue involving loss and/or apoptosis of cells ofthe tissue which comprises administering to the subject a compositioncomprising an amount of an agent which induces phosphorylation and/oractivation of protein kinase B, the composition being administered in anamount effective to cause proliferation of the cells and/or inhibitapoptosis of the cells of the tissue within the subject so as to therebytreat the disorder.

This invention also provides a method of treating a subject sufferingfrom a disorder of a tissue involving loss and/or apoptosis of cells ofthe tissue which comprises administering to the subject a compositioncomprising an amount of an agent which induces phosphorylation and/oractivation of an extracellular signal-regulated protein kinase, thecomposition being administered in an amount effective to inhibitapoptosis and/or cause proliferation of the cells of the tissue withinthe subject so as to thereby treat the disorder.

This invention also provides a method of treating a subject sufferingfrom a disorder of a tissue involving loss and/or apoptosis of cells ofthe tissue which comprises administering to the subject a compositioncomprising an amount of an agent which induces activation of CXCR4, thecomposition being administered in an amount effective to causeproliferation of the cells and/or inhibit apoptosis of the cells of thetissue within the subject so as to thereby treat the disorder.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D: IL-8/Gro-Alpha CXC Chemokines Regulate Migration Of HumanEndothelial progenitor cells (angioblasts) To Myocardial Tissue In VivoAnd Subsequent Development Of Vasculogenesis.

(A) DiI-labelled human endothelial progenitor cells (angioblasts) (>98%CD34+ purity) injected intravenously into nude rats infiltrate ratmyocardium after coronary artery ligation and infarction but not aftersham operation at 48 hours.

(B) Migration of human endothelial progenitor cells (angioblasts) toischemic rat myocardium is inhibited by mAbs against either rat IL-8 orthe IL-8/Gro-alpha chemokine family receptors CXCR1 and CXCR2 (allp<0.01), but not against VEGF or its receptor Flk-1 (results areexpressed as mean+sem of three separate experiments).

(C) Masson's trichrome stain of rat myocardial infarct bed at two weeksafter LAD ligation demonstrating diffuse increase in matrix depositionand few capillaries in representative animal injected with saline(×400), diffuse increase in capillaries (arrowheads) and reduction inmatrix deposition in representative animal injected with human bonemarrow-derived endothelial progenitor cells (×400), and reduction incapillary numbers in representative animal injected with humanendothelial progenitor cells (angioblasts) together with mAb againsthuman CXCR1/2 (×400).

(D) Intracardiac injection of IL-8 or SDF-1 at 1 μg/ml significantlyincreases in vivo chemotaxis of DiI-labelled human endothelialprogenitor cells (angioblasts)(98% CD34+ purity) into non-ischemic ratheart in comparison with injection of saline or stem cell factor (SCF),p<0.01 (results are expressed as mean+sem of three separateexperiments). Below is shown representative fluorescence microscopy ofintravenously-injected DiI-labelled human endothelial progenitor cellsinfiltrating non-ischemic rat heart after intracardiac injection withsaline, IL-8 or SDF-1.

FIGS. 2A-2C: Blocking CXCR4/SDF-1 Interactions Redirects IntravenouslyInjected Human Endothelial progenitor cells From Bone Marrow To IschemicMyocardium.

(A) the proportion of human CD34+CD117^(bright) endothelial progenitorcells (angioblasts) in rat bone marrow 2-14 days after intravenousinjection is significantly increased following ischemia induced by LADligation (results are expressed as mean+sem of bone marrow studies inthree animals at each time point).

(B) and (C) depict the effects of mAbs against CXCR4, SDF-1 or CD34 ontrafficking of human CD34+ endothelial progenitor cells (angioblasts) torat bone marrow and myocardium following LAD ligation. Co-administrationof anti-CXCR4 or anti-SDF-1 significantly reduced trafficking ofintravenously injected human CD34+ cells to rat bone marrow at 48 hoursand increased trafficking to ischemic myocardium, whereas anti-CD34 mAbhad no effect (results are expressed as mean+sem of bone marrow andcardiac studies performed in three LAD-ligated animals at 48 hours afterinjection).

FIGS. 3A-3F. Redirected Trafficking Of Human Endothelial ProgenitorCells (angioblasts) To The Site Of Infarction Induces Vasculogenesis AndProtects Cardiomyocytes Against Apoptosis.

(A) Myocardial infarct bed two weeks post-LAD ligation fromrepresentative animals in each group stained with Masson's trichrome(upper panel) or immunoperoxidase after binding of anti-CD31 mAb (lowerpanel). The infarct zones of rats receiving either 10³ or 10⁵endothelial progenitor cells (angioblasts) show myocardial scarscomposed of paucicellular, dense fibrous tissue stained blue bytrichrome (×400). In contrast, the infarct zones of rats injected with10⁵ endothelial progenitor cells plus anti-CXCR4 mAb show significantincrease in cellularity of granulation tissue, minimal matrix depositionand fibrosis, and numerous medium-sized capillaries of human origin. Theinfarct zones of rats injected with 2×10⁵ endothelial progenitor cellsshow a similar reduction in fibrous tissue and increase in medium-sizedcapillaries, and an additional increase in large-sized vessels of humanorigin.

(B) and (C) show the relationship between the number of humanCD117^(bright) endothelial progenitor cells injected intravenously (10³,10⁵, 10⁵ plus anti-CXCR4 mAb, and 2×10⁵) and development of rat infarctbed vasculogenesis at two weeks, defined as the mean number ofcapillaries/high power field (hpf) with medium- or large-sized lumendiameter (respectively, 0.02 mm mean diameter with 3-6 contiguousendothelial lining cells and 0.05 mm mean diameter with >6 contiguousendothelial lining cells). Results are expressed as the mean+sem of atleast 15 hpf in three separate experiments.

(B) the groups receiving either 2×10⁵ endothelial progenitor cells(angioblasts) or 10⁵ endothelial progenitor cells plus anti-CXCR4 mAbdemonstrated 1.7-fold higher numbers of medium-sized capillariescompared with the other two groups (p<0.01).

(C) the group receiving 2×10⁵ endothelial progenitor cells (angioblasts)additionally demonstrated 3.3-fold higher numbers of large-lumencapillaries compared with the groups receiving 10³ or 10⁵ endothelialprogenitor cells (p_(<)0.01), and 2-fold higher numbers of large-lumencapillaries compared with the group receiving 10⁵ endothelial progenitorcells plus anti-CXCR4 mAb (p<0.01).

(D) shows that co-administration of anti-CXCR4 mAb together with thehighest concentration of endothelial progenitor cells (angioblasts),2×10⁵, resulted in a further 23% increase in growth of large-lumencapillaries. More strikingly, there was a further 2-fold increase incapillary numbers when 2×10⁵ endothelial progenitor cells were injectedintravenously after direct intracardiac delivery of 1.0 μg/ml SDF-1 intoinfarcted hearts (p<0.01) (results are expressed as mean+sem of threeseparate experiments).

(E) shows that at 2 weeks the numbers of apoptotic myocytes at theperi-infarct rim, defined by concomitant staining with anti-desmin mAband DNA end-labeling using TUNEL technique, are significantly reduced inrats receiving either 2.0×10⁵ endothelial progenitor cells or 10⁵endothelial progenitor cells together with anti-CXCR4 mAb in comparisonto rats receiving 10³ or 10⁵ endothelial progenitor cells (p<0.01)(results are expressed as mean+sem of three separate experiments).

(F) shows that co-administration of anti-CXCR4 mAb or intracardiacinjection of SDF-1 resulted in further reductions in cardiomyocyteapoptosis of 65% and 76%, respectively, at two weeks (both p<0.001)(results are expressed as mean+sem of three separate experiments).

FIGS. 4A-4H. Infarct Bed Vasculogenesis Improves Long-Term MyocardialFunction Through Mechanisms Involving Both Cardiomyocyte Protection andProliferation/Regeneration.

(A) and (B) show the relationship between the number of humanCD117^(bright) endothelial progenitor cells (angioblasts) injectedintravenously (10³, 10⁵, 10⁵ plus anti-CXCR4 mAb, and 2×10⁵) andimprovement in myocardial function at 15 weeks, defined as meanimprovement in left ventricular ejection fraction (LVEF) (A) and meanreduction in left ventricular area at end-systole (LVAs) (B). Nosignificant improvement in these parameters was observed in the groupsreceiving 10³ or 10⁵ endothelial progenitor cells in comparison to ratsreceiving saline alone. In contrast, rats receiving 10⁵ endothelialprogenitor cells plus anti-CXCR4 mAb demonstrated significant recoveryin LVEF and reduction in LVAs (both p<0.001). The group receiving 2×10⁵endothelial progenitor cells demonstrated still 50% greater recovery inLVEF and reduction in LVAs (both p<0.001).

(C) Section from infarct of representative animal receiving 2×10⁵endothelial progenitor cells (angioblasts) showing a high frequency ofcardiomyocytes staining positively for both cardiac-specific troponin Iand rat-specific Ki-67 (arrows). Note the proximity of Ki-67-positivecardiomyocytes to capillaries (arrowheads).

(D) Section from infarct of representative animal receiving 2×10⁵endothelial progenitor cells showing “finger” of cardiomyocytes of ratorigin, as determined by expression of rat MHC class I molecules,extending from the peri-infarct region into the infarct zone. Thesecellular islands contain a high frequency of myocytes stainingpositively for both cardiac-specific troponin I and rat-specific Ki-67(arrows). Sections from infarcts of representative animals receivingsaline do not show same frequency of dual staining myocytes. Bar graphshows that the group of animals receiving 2×10⁵ human endothelialprogenitor cells has a significantly higher index of cell cyclingcardiomyocytes at the peri-infarct region than saline controls or shamoperated animals (both p<0.01). No difference between the groups is seenat sites distal to the infarct.

(E) shows that the index of cell-cycling cardiomyocytes at theperi-infarct rim was increased by a further 1.9-fold when 2×10⁵ humanendothelial progenitor cells were intravenously co-administered togetherwith SDF-1 injected directly into the ischemic myocardium alone(p<0.01), or an 8-fold cumulative increase in cell-cyclingcardiomyocytes at two weeks compared with LAD-ligated controls receivingsaline (results are expressed as mean+sem of three separateexperiments).

(F) shows that intravenous co-administration of anti-CXCR4 mAb, orintracardiac co-administration of SDF-1, but not IL-8, results in 2.8 to4-fold greater LVEF improvement, determined by echocardiography,compared with intravenous injection of 2×10⁵ endothelial progenitorcells alone (p<0.01) (results are expressed as mean+sem of threeseparate experiments).

(G) shows that at 15 weeks the mean proportion of scar/normal leftventricular myocardium in rats receiving either or 10⁵ endothelialprogenitor cells together with anti-CXCR4 mAb was significantly reducedin comparison to rats receiving either 10³ or 10⁵ endothelial progenitorcells (angioblasts) alone, or saline (p<0.01). The group receiving2.0×10⁵ endothelial progenitor cells demonstrated still 38% greaterreduction in the ratio of scar/muscle tissue (results are expressed asmean+sem of three separate experiments).

(H) Sections of rat hearts stained with Masson's trichrome at 15 weeksafter LAD ligation and injection of 2.0×10⁶ G-CSF mobilized human cellscontaining 10³ (left) or 2.0×10⁵ (right) CD117^(bright) endothelialprogenitor cells. Hearts of rats receiving 10³ endothelial progenitorcells had greater loss of anterior wall mass, collagen deposition(lighter gray), and septal hypertrophy compared with hearts of ratsreceiving 2.0×10⁵ endothelial progenitor cells.

FIG. 5: This figure shows mRNA expression of three genes in the ischemicrat hearts at various time points. You will see that at 48 hours and 2weeks after LAD ligation and ischemia, HBP23 (the rat homologue of humanPAG/NKEF-A,B,C, all of which are part of the family of peroxiredoxins(Prx)) is decreased, and vitamin D3 upregulated protein VDUP-1 isincreased. The early (48 hour) reduction in PRX and increase in VDUP-1results in a compensatory increase in thiol reductase thioredoxin (TRX).Note that endothelial progenitor cell therapy reverses this pattern ofmRNA expression.

FIG. 6: DNA sequence (SEQ ID NO:1) corresponding to mRNA encodingVDUP-1.

FIG. 7: This figure shows catalytic DNA 5′-AT-3′ cleavage sites onVDUP-1 DNA (SEQ ID NO:3) corresponding to the coding region of VDUP-1mRNA. Cleavage site pairs are uppercase.

FIG. 8: This figure shows catalytic DNA 5′-GC-3′ cleavage sites onVDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNA. Cleavage sitepairs are uppercase.

FIG. 9: This figure shows catalytic DNA 5′-GT-3′ cleavage sites onVDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNA. Cleavage sitepairs are uppercase.

FIG. 10: This figure shows catalytic DNA 5′-AC-3′ cleavage sites onVDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNA. Cleavage sitepairs are uppercase.

FIG. 11: This figure shows sites which can be cleaved by a hammerheadribozyme in VDUP-1 DNA (SEQ ID NO:3) corresponding to the VDUP-1 mRNAcoding region. Uppercase “T” represents cleavage site.

FIG. 12: This figure shows that at VDUP-1 enzyme concentrations rangingfrom 0.05 μM to 5 μM the sequence-specific VDUP1 DNA enzyme cleaved asynthetic rat VDUP1 oligonucleotide in a concentration- andtime-dependent manner.

FIG. 13: (a) Shows intramyocardial injection of the ratsequence-specific VDUP1 DNA enzyme at 48 hours after LAD ligationresulted in a 75% mean inhibition of proliferating cardiac fibroblastsin the infarct zone two weeks later in comparison to injection ofscrambled DNA enzyme control (p<0.01). (b) Shows injection of VDUP1 DNAenzyme resulted in 20% mean reduction in apoptotic cardiomyocytes at theperi-infarct region relative to injection with the scrambled. DNA enzymecontrol (p<0.05).

FIG. 14: (a) Shows inhibition of fibroblast proliferation andcardiomyocyte apoptosis resulted in significant reduction of mature scardeposition in the infarct zone, from a mean of 35% for animals receivingcontrol scrambled DNA enzyme to a mean of 20% for those receiving VDUP1DNA enzyme (p<0.01). (b) Shows animals receiving VDUP1 DNA enzymedemonstrated a 50% mean recovery in cardiac function, as determined byejection fraction, whereas no improvement was seen in animals receivingscrambled control DNA enzyme (p<0.01)

FIG. 15: (a) Shows that G-CSF, when used systematically at the samedosages after myocardial infarction, is a more potent inducer of cardiacneovascularization than GM-CSF. (B) Shows G-CSF is a more potentinhibitor of cardiomyocyte apoptosis than GM-CSF.

FIG. 16: (a) Shows G-CSF is a more potent inducer of cardiomyocyteregeneration than GM-CSF, and (b) shows that G-CSF enables significantlygreater recovery of cardiac function after acute myocardial infarction.

FIG. 17: (a) Shows intravenous administration of anti-CXCR4 monoclonalantibody after acute myocardial infarction induces significantly greaternumbers of blood vessels at the peri-infarct region. (b) Shows thegreater recovery of cardiac/myocardial function than with controlantibodies.

FIG. 18: Shows myocardial expression of SDF-1 mRNA increased by twoweeks, but not within the first 48 hours after myocardial infarction,and is inhibited by subcutaneously injecting GM-CSF.

FIG. 19: (a) Shows intramyocardial injection of SDF-1 inducesneovascularization at the peri-infarct region and (b) protectscardiomyocytes at the peri-infarct region against apoptosis.

FIG. 20: (a) Shows intramyocardial administration of SDF-1 alone, andsynergistically with GM-CSF induces cardiomyocyte regeneration at theperi-infarct region, and (b) improves cardiac function.

FIG. 21: (a) Shows Human angioblasts, or endothelial progenitor cells,induce neovascularization as early as 5 days post-infarct. (b) ShowsHuman angioblasts, or endothelial progenitor cells protect peri-infarctcardiomyocytes against apoptosis as early as 5 days post infarct.

FIG. 22: (a) Shows animals receiving human bone marrow-derived CD34+cells demonstrated numerous clusters of small, cycling cells at theperi-infarct region that were of rat origin, as defined by a monoclonalantibody specific for rat Ki67. (b) Shows tissues obtained from animalssacrificed at two weeks after human CD34+ administration no longerdemonstrated clusters of small, cycling cardiomyocyte progenitors, butinstead a high frequency of large, mature rat cardiomyocytes at theperi-infarct region with detectable DNA activity, as determined by dualstaining with mAbs reactive against cardiomyocyte-specific troponin Iand mAbs reactive against rat Ki67. (c) Shows cycling by the samestaining.

FIG. 23: Shows by RT-PCR, HBP23 mRNA levels in rat hearts decreased attwo weeks post-LAD ligation by a mean of 34% compared with normal rathearts.

FIG. 24: (a) Shows a DNA enzyme against HBP23 cleaved the 23-baseoligonucleotide synthesized from the sequence of rat HBP23 mRNA, in adose- and time-dependent manner. (b) Shows densitometric analysis ofRT-PCR products following reverse transcription of cellular mRNA,demonstrating the HBP23 DNA enzyme inhibited steady-state mRNA levels incultured rat cells by over 80% relative to the scrambled DNA.

FIG. 25: (a) Shows HBP23 DNA enzyme had no effect on induction ofneovascularization by human bone marrow angioblasts, no increasedmyocardial capillary density in comparison to saline controls is seen.(b) Shows injection of the HBP23 DNA enzyme, but not the scrambledcontrol, abrogated the anti-apoptotic effects of neovascularization. (c)Shows injection of the HBP23 DNA enzyme, but not the scrambled control,abrogated the improvement in cardiac function.

FIG. 26: (a) This figure shows the pattern of CXCR4 expression followingacute myocardial ischemia is focal and peri-infarct. (b) Myocardialinfarct bed two weeks post myocardial infarction from a representativeanimal shows CXCR4 staining in dark gray expressed in peri-infarctcardiomyocytes.

FIG. 27: Single myocardial injection of SDF-1 after acute ischemiainduces early phosphorylation of potein kinase B (AKT). SDF-1intramyocardial administration (4 ug/kg) at time of infarction resultsin significant early phosphorylation of AKT at 24 hours post-myocardialinfarction compared to animals receiving saline controls. This effectwas not measurable at two weeks post-myocardial infarction.

FIG. 28: Cultured rat neonatal cardiomyocytes express CXCR4. Culturedrat neonatal cardiomyocytes in vitro highly express CXCR4 throughout thecytoplasm as defined by immunohistochemical staining using a mAb thatcross reacts with rat CXCR4 (dark gray in ×200 and ×400).

FIG. 29: SDF-1 induces phosphorylation of AKT and ERK in rat neonatalcariomyocytes in time dependent manner. The left hand panel showsoptimal phosphorylation of AKT as early as five minutes when culturedrat neonatal cardiomyocytes are cultured with 100 nM of SDF-1. Righthand panel similarly demonstrates maximal ERK phosphorylation at tenminutes as defined by western blot analysis.

FIG. 31: Intracardiac administration of SDF-1 augments CSF-inducedneovascularization and regeneration following acute ischemia. The lefthand panel shows that the combination of subcutaneous G-CSF andintracardiac SDF-1 administration results in slightly higher numbers oflarge diameter blood vessels as defined by having greater than 6 nucleiper high power field. The right hand panel shows similar ratios areobserved with the number of cardiomyocytes entering cell cycle asdefined by KI67 and troponin I co-staining at two weeks post myocardialinfarction.

FIG. 32: Intracardiac administration of SDF-1 augments CSF-inducedfunctional myocardial recovery following myocardial ischemia. Thecombination of subcutaneous G-CSF and intracardiac SDF-1 administrationresults in over a 35% improvement in ejection fraction as defined byM-mode echocardiography at two weeks post myocardial infarction. Incontrast animals receiving saline or only of subcutaneous GM-CSF had anadditional regression in cardiac functional capacity.

FIG. 30: SDF-1 protects rat neonatal cardiomyocytes against H₂O₂-inducedapoptosis in a dose dependent manner. The left hand panel demonstratesthe protective effect of SDF-1 using the highest concentration (10 μM).The right hand panel demonstrates at the highest concentration of H₂O₂the protective effects of SDF-1 on cardiomyocyte apoptotic deathfollowing H₂O₂ stimulation is dose dependent with 10 μM having maximaleffects. Medium=no SDF.

DETAILED DESCRIPTION

As used herein, “VEGF” is defined as vascular endothelial growth factor.“VEGF-R” is defined as vascular endothelial growth factor receptor.“FGF” is defined as fibroblast growth factor. “IGF” is defined asInsulin-like growth factor. “SCF” is defined as stem cell factor.“G-CSF” is defined as granulocyte colony stimulating factor. “M-CSF” isdefined as macrophage colony stimulating factor. “GM-CSF” is defined asgranulocyte-macrophage colony stimulating factor. “MAPK” ismitogen-activated protein kinase. “MCP” is defined as monocytechemoattractant protein. “AKT” is a serine/threonine kinase also knownas protein kinase B (PKB). “ERK” is extracellular signal-regulatedprotein kinase.

As used herein “angioblasts” is synonymous with the term “endothelialprogenitor cells”.

As used herein, “CXC” chemokine refers to the structure of thechemokine. Each “C” represents a cysteine and “X” represents any aminoacid. “CXCR4” refers to CXC receptor 4.

As used herein, “CC” chemokine refers to the structure of the chemokine.Each “C” represents a cysteine.

A “cardiac progenitor cell” refers to a cell that is resident in theheart, or that comes into the heart from elsewhere after acute ischemia,is smaller than mature cardiomyocytes, expresses alpha sarcomeric actinbut is negative for troponin, is normally quiescent but can be inducedto go into cell cycle as defined by positive Ki67 staining. “Cardiacprogenitor cell” may be used synonymously with “cardiomyocyte progenitorcell”.

“Catalytic” shall mean the functioning of an agent as a catalyst, i.e.an agent that increases the rate of a chemical reaction without itselfundergoing a permanent structural change.

“Nucleic acid” shall include without limitation any nucleic acid,including, without limitation, DNA, RNA, oligonucleotides, orpolynucleotides, and analogs or derivatives thereof. The nucleotidesthat form the nucleic acid may be nucleotide analogs or derivativesthereof. The nucleic acid may incorporate non nucleotides.

“Nucleotides” shall include without limitation nucleotides and analogsor derivatives thereof. For example, nucleotides may comprise the basesA, C, G, T and U, as well as derivatives thereof. Derivatives of thesebases are well known in the art, and are exemplified in PCR Systems,Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, RocheMolecular Systems, Inc., Branchburg, N.J., USA).

“Proliferation” and “regeneration” are used synonymously when referringto cardiomyocytes in this application. “Proliferation” in respect tocardiomyocytes, shall mean a fold increase in proportion ofcardiomyocytes entering the cell cycle relative to untreated rat heart.

“Systemic” administration includes, not is not limited to,intramuslcular, sub-cutaneous, intravascular, and intraperitonealadministration. In other forms of administration, each and any of theagents may be administered intracoronarily, intramyocardially, and bystent, scaffold or slow release formation also.

“Tissue” includes heart tissue and, in that embodiment, “cells” includecardiomyocytes. “Tissue” may also include lung, brain, gastrointestinal,liver, kidney and other tissues. “Cells” also includes stem cells whichcan differentiate into the cell type being lost or progenitors of thecell type being lost in the disorder of the tissue.

“Trafficking” means the blood-borne migration of cells, in particularangioblast/endothelial progenitor cells.

This invention provides a method of treating a disorder of a subject'sheart involving loss of cardiomyocytes which comprises administering tothe subject a composition comprising an amount of a humanstromal-derived factor-1 and an amount of a human granulocyte-colonystimulating factor, the composition being administered in an amounteffective to cause proliferation of cardiomyocytes within the subject'sheart so as to thereby treat the disorder.

This invention further provides the instant method, wherein the humanstromal-derived factor-1 is human stromal-derived factor-1α, humanstromal-derived factor-1β, or human stromal-derived factor-ly.

This invention further provides the instant method, wherein the tissueis heart tissue and the cells are cardiomyocytes. This invention furtherprovides the instant method, wherein the disorder comprises myocardialinfarction, congestive heart failure, chronic ischemia, or ischemicdisease.

This invention further provides the instant method, further comprisingadministering to the subject an amount of one or more of a humangranulocyte macrophage-colony stimulating factor, a human interleukin-8,a human vascular endothelial growth factor, a human fibroblast growthfactor, a human Gro family chemokine, human endothelial progenitorcells, or a pro-angiogenic agent, the amount, or if appropriate amounts,thereof being effective to cause proliferation of cardiomyocytes withinthe subject's heart so as to thereby treat the disorder.

This invention further provides the instant method, wherein thecomposition is administered intramyocardially, intracoronarily, via astent, a scaffold, or a slow-release formulation.

This invention also provides a method of treating a subject sufferingfrom a disorder of a tissue involving loss and/or apoptosis of cells ofthe tissue which comprises administering to the subject a compositioncomprising an amount of an agent which induces phosphorylation and/oractivation of protein kinase B, the composition being administered in anamount effective to cause proliferation of the cells and/or inhibitapoptosis of the cells of the tissue within the subject so as to therebytreat the disorder.

This invention further provides the instant method, wherein the agent ishuman human stromal-derived factor-la, human stromal-derived factor-1β,or human stromal-derived factor-1γ.

This invention further provides the instant method, wherein the tissueis heart tissue and the cells are cardiomyocytes. This invention furtherprovides the instant method, wherein the disorder from which the subjectis suffering comprises myocardial infarction, congestive heart failure,chronic ischemia, or ischemic disease.

This invention further provides the instant method, wherein the tissueis heart tissue and the cells are progenitors of cardiomyocytes or stemcells that differentiate to cardiomyocytes.

This invention further provides the instant method, wherein the tissueis striated muscle, liver, kidney, neuronal or gastrointestinal tissue.

This invention further provides the instant method, wherein the agent isinsulin, endothelin-1, urocrotin, cardiotropin-1, erythropoietin,leukemia inhibitory factor-1, tumor necrosis factor-alpha.

This invention further provides the instant method, further comprisingadministering an amount of one or more of a human granulocyte-colonystimulating factor, a human stromal-derived factor-1, a humangranulocyte macrophage-colony stimulating factor, a human interleukin-8,a human vascular endothelial growth factor, a human fibroblast growthfactor, a human Gro family chemokine, human endothelial progenitorcells, or a pro-angiogenic agent, the amount, or if appropriate amounts,effective to cause proliferation of the cells and/or inhibit apoptosisof the cells of the tissue of the subject so as to thereby treat thedisorder.

This invention further provides the instant method, wherein thecomposition is administered intramyocardially, intracoronarily, via astent, a scaffold, a slow-release formulation, intramuscularly,intravenously, intra-arterially, or sub-cutaneously.

This invention also provides a composition comprising a humanstromal-derived factor-1 and a human granulocyte-colony stimulatingfactor.

This invention also provides a method of treating a subject sufferingfrom a disorder of a tissue involving loss and/or apoptosis of cells ofthe tissue which comprises administering to the subject a compositioncomprising an amount of an agent which induces phosphorylation and/oractivation of an extracellular signal-regulated protein kinase, thecomposition being administered in an amount effective to inhibitapoptosis and/or cause proliferation of the cells of the tissue withinthe subject so as to thereby treat the disorder.

This invention further provides the instant method, wherein the agent ishuman human stromal-derived factor-la, human stromal-derived factor-1β,or human stromal-derived factor-1γ.

This invention further provides the instant method, wherein the tissueis heart tissue and the cells are cardiomyocytes. This invention furtherprovides the instant method, wherein the disorder from which the subjectis suffering comprises myocardial infarction, congestive heart failure,chronic ischemia, or ischemic disease.

This invention further provides the instant method, wherein the tissueis heart tissue and the cells are progenitors of cardiomyocytes or stemcells that differentiate to cardiomyocytes.

This invention further provides the instant method, further comprisingadministering an amount of one or more of a human granulocyte-colonystimulating factor, a human stromal-derived factor-1, a humangranulocyte macrophage-colony stimulating factor, a human interleukin-8,a human vascular endothelial growth factor, a human fibroblast growthfactor, a human Gro family chemokine, human endothelial progenitorcells, an activator of protein kinase B, or a pro-angiogenic agent, theamount, or if appropriate amounts, thereof being effective to inhibitapoptosis and/or cause proliferation of the cells of the tissue withinthe subject so as to thereby treat the disorder.

This invention further provides the instant method, wherein the agent isadministered intramyocardially, intracoronarily, via a stent, ascaffold, or a slow-release formulation, intramuscularly, intravenously,intra-arterially, or sub-cutaneously.

This invention also provides the method of treating a subject sufferingfrom a disorder of a tissue involving loss and/or apoptosis of cells ofthe tissue which comprises administering to the subject a compositioncomprising an amount of an agent which induces activation of CXCR4, thecomposition being administered in an amount effective to causeproliferation of the cells and/or inhibit apoptosis of the cells of thetissue within the subject so as to thereby treat the disorder.

This invention further provides the instant method, wherein the tissueis heart tissue and the cells are cardiomyocytes. This invention furtherprovides the instant method, wherein the agent is administeredintramyocardially or intracoronarily via a stent, a scaffold, or aslow-release formulation.

This invention further provides the instant method, wherein the agent isadministered systemically.

This invention further provides the use of an amount of a humanstromal-derived factor-1 and an amount of a human granulocyte-colonystimulating factor for the manufacture of a composition for treating adisorder of a subject's heart involving loss of cardiomyocytes.

This invention further provides the use of an agent which inducesphosphorylation and/or activation of protein kinase B for themanufacture of a composition for treating a disorder of a subject'stissue involving loss of the cells of the tissue.

This invention further provides the use of an amount of an agent whichinduces phosphorylation and/or activation of extracellular signalregulated protein kinase for the manufacture of a composition fortreating a disorder of a subject's tissue involving loss of the cells ofthe tissue.

This invention further provides the use of an amount of an agent whichinduces activation of CXCR4 for the manufacture of a composition fortreating a disorder of a subject's tissue involving loss of cells of thetissue.

This invention provides a method of treating a disorder of a subject'sheart involving loss of cardiomyocytes which comprises administering tothe subject an amount of an agent effective to cause cardiomyocyteproliferation within the subject's heart so as to thereby treat thedisorder.

In one embodiment the agent is human endothelial progenitor cells. Inone embodiment the endothelial progenitor cells are bone marrow-derived.In another they are derived from cord blood, or embryonic or fetalsources. Effective amounts of endothelial progenitor cells sufficient tocause cardiomyocyte proliferation can be done based on animal data usingroutine computational methods. In one embodiment the effective amount isabout 1.5×10⁵ endothelial progenitor cells per kg body mass to about3×10⁵ per kg body mass. In another embodiment the effective amount isabout 3×10⁵ per kg body mass to about 4.5×10⁵ endothelial progenitorcells per kg body mass. In another embodiment the effective amount isabout 4.5×10⁵ per kg body mass to about 5.5×10⁵ endothelial progenitorcells per kg body mass. In another embodiment the effective amount isabout 5.5×10⁵ per kg body mass to about 7×10⁵ endothelial progenitorcells per kg body mass. In another embodiment the effective amount isabout 7×10⁵ per kg body mass to about 1×10⁶ endothelial progenitor cellsper kg body mass. In another embodiment the effective amount is about1×10⁶ per kg body mass to about 1.5×10⁵ endothelial progenitor cells perkg body mass. In one embodiment the effective amount of humanendothelial progenitor cells is between about 1.5×10⁵ and 4.5×10⁵endothelial progenitor cells per kg of the subject's body mass and in apreferred embodiment the effective amount is about 5×10⁵ endothelialprogenitor cells per kg of the subject's body mass.

In one embodiment the endothelial progenitor cells are allogeneic withrespect to the subject. In differing embodiments the subject is an adultor an embryo or a fetus. In another embodiment the endothelialprogenitor cells are derived from cloned autologous embryonic stemcells.

In one embodiment the agent induces expression of a mRNA encoding aperoxiredoxin. The expression of peroxiredoxin mRNA may be increased,for example, by administration of 2(3)-t-butyl-4-hydroxyanisole (BHA)(see 106) which has been shown to increase expression of Peroxiredoxin-1when administered by diet. Alternatively, local control of 0₂ can havethe same effect (see 107). Peroxiredoxin mRNA expression, such as thatof thiol peroxidases, may also be induced by heme, cadmium or cobalt(see 108). Peroxiredoxins include, but are not limited to, PAG, HBP23,MSP23, NKEF.

In another embodiment the agent induces expression of a mRNA encodingNF-E2-related factor 2 (Nrf2). Cytomplasmic NF-E2-related factor 2(Nrf2) expression can be indirectly increased by raising free Nrf2levels. Since Nrf2 is tightly bound to keap1 in the cytoplasm thenreducing expression of keap1 mRNA is a suitable target e.g. by keap1antisense oligonucleotides or catalytic nucleic acids (see 109 forkeap1). Also, blocking the interaction between Nrf2 and Keap1 byinhibiting the interaction of the Neh2 domain of Nrf2 and the DGR domainof keap1, e.g. by using the entire Neh2 domain of nrf2, amino acids1-73, or only the hydrophilic region, amino acids 33-73 (see 109) willincrease free cytoplasmic Nrf-2. In another embodiment the agent inducesdissociation of a Nrf2 protein from a Keap-1. In another embodiment theagent inhibits association of a Nrf2 protein with a Keap-1. In anotherembodiment the agent inhibits association of a thiol reductasethioredoxin with a VDUP-1 protein. In another embodiment the agentinhibits c-Abl tyrosine kinase activation. In a further embodiment theagent is STI-571.

In one embodiment the agent is a CXC chemokine. In further embodimentsthe agent is Stromal-Derived Factor-1, 11-8 or Gro-Alpha. In oneembodiment the amount of CXC chemokine administered is between 0.2 and 5μg/ml at a max volume of 10 ml for a 70 kg human subject. In a preferredembodiment the amount is about 1 μg/ml. In one embodiment the agent isan inhibitor of plasminogen activator inhibitor-1. In another embodimentthe agent is an antibody directed against an epitope of CXCR4. In oneembodiment the amount of antibody directed against an epitope of CXCR4is between 25 and 75 μg/ml, at a max volume of 10 ml for a 70 kg humansubject. A simple calculation is performed for subjects of differentmass. In a preferred embodiment the amount is about 50 μg/ml.

In differing embodiments the agent is Stromal-Derived Factor-1 alpha orStromal-Derived Factor-1 beta. In differing embodiments the chemokinecan be administered intramyocardially, intracoronary, and/or via astent, a scaffold, or as a slow-release formulation.

In differing embodiments the agent is G-CSF, GM-CSF, or a Gro familychemokine. In a further embodiment the agent is Gro alpha.

SDF variants may be employed in the present invention. For example, amutein, an analog, fusion protein, functional derivative, isoform,allelic variant or effective fragment of SDF. The amino acid sequence ofa number of different native mammalian SDF-1 alpha, beta and gammaproteins are known, including human, rat, mouse, and cat. See, e. g.,Shirozu et al., Genomics, 28: 495, 1995; Tashiro et al., Science 261:600, 1993; Nishimura et al., Eur. J. Immunogenet. 25: 303, 1998; andGenBank Accession No. AF189724. A preferred form of SDF-1 protein is apurified native SDF-1 protein that has an amino acid sequence identicalto one of the foregoing mammalian SDF-1 proteins. Variants of nativemammalian SDF-1 proteins such as fragments, analogs and derivatives ofnative mammalian SDF-1 proteins may also be used in the invention. Suchvariants include, e. g., a polypeptide encoded by a naturally occurringallelic variant of native SDF-1 gene (i. e., a naturally occurringnucleic acid that encodes a naturally occurring mammalian SDF-1protein), a polypeptide encoded by an alternative splice form of anative SDF-1 gene, a polypeptide encoded by a homolog of a native SDF-1gene, and a polypeptide encoded by a non-naturally occurring variant ofa native SDF-1 gene. SDF-1 protein variants have a peptide sequence thatdiffers from a native SDF-1 protein in one or more amino acids. Thepeptide sequence of such variants can feature a deletion, addition, orsubstitution of one or more amino acids of a native SDF-1 protein. Aminoacid insertions are preferably of about 1 to 4 contiguous amino acids,and deletions are preferably of about 1 to 10 contiguous amino acids. Insome applications, variant SDF-1 proteins substantially maintain anative SDF-1 protein functional activity (e.g., the ability to causecellular chemotaxis). For other applications, variant SDF-1 proteinslack or feature a significant reduction in a SDF-1 protein functionalactivity. Where it is desired to retain a functional activity of nativeSDF-1 protein, preferred SDF-1 protein variants can be made byexpressing nucleic acid molecules within the invention that featuresilent or conservative changes. Variant SDF-1 proteins with substantialchanges in functional activity can be made by expressing nucleic acidmolecules within the invention that feature less than conservativechanges.

SDF-1 protein fragments corresponding to one or more particular motifsand/or domains or to arbitrary sizes, for example, at least 5, 10, 25,50, or 75 amino acids in length are within the scope of the presentinvention. Isolated peptidyl portions of SDF-1 proteins can be obtainedby screening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a SDF-1 protein of the present invention may bearbitrarily divided into fragments of desired length with no overlap, ofthe fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of native SDF-1protein. Another aspect of the present invention concerns recombinantforms of the SDF-1 proteins. Recombinant polypeptides preferred by thepresent invention, in addition to a native SDF-1 protein, are encoded bya nucleic acid that has at least 85% sequence identity (e.g., 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) with thenucleic acid sequence of a gene encoding a mammalian SDF-1 protein. In apreferred embodiment, variant SDF-1 proteins have one or more functionalactivities of native SDF-1 protein.

SDF-1 protein variants can be generated through various techniques knownin the art. For example, SDF-1 protein variants can be made bymutagenesis, such as by introducing discrete point mutation (s), or bytruncation. Mutation can give rise to a SDF-1 protein variant havingsubstantially the same, or merely a subset of the functional activity ofnative SDF-1 protein. Alternatively, antagonistic forms of the proteincan be generated which are able to inhibit the function of a naturallyoccurring form of the protein, such as by competitively binding toanother molecule that interacts with a SDF-1 protein. In addition,agonistic forms of the protein may be generated that constitutivelyexpress on or more of the functional activities of a native SDF-1protein. Other SDF-1 protein variants that can be generated includethose that are resistant to proteolytic cleavage, as for example, due tomutations which alter protease target sequences. Whether a change in theamino acid sequence of a peptide results in a variant having one or morefunctional activities of a native SDF-1 protein can be readilydetermined by testing the variant for a native SDF-1 protein functionalactivity.

As another example, SDF-1 protein variants can be generated from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be carried out in an automatic DNA synthesizer, andthe synthetic genes then ligated into an appropriate expression vector.The purpose of a degenerate set of genes is to provide, in one mixture,all of the sequences encoding the desired set of potential SDF-1 proteinsequences. The synthesis of degenerate oligonucleotides is well known inthe art (see for example, Narang, S A (1983) Tetrahedron 39: 3; Itakuraet al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos.Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakuraet al.

As used herein “a human stromal-derived factor-1” means a polypeptidewhich has the same or substantially the same amino acid sequence andbiological activity as a naturally occurring human stromal-derivedfactor-1, including specifically, any of human stromal-derivedfactor-1α, 1β, or 1γ. The term “human stromal-derived factor-1” thusencompasses polypeptides which have one or more additional amino acids,typically less than 5 additional amino acids at either the N-terminus orthe C-terminus or both so long as the biological activity is retained,and specifically includes a polypeptide having an N-terminal methionineadded to the sequence present in a naturally occurring stromal-derivedfactor-1. The term also encompasses conjugates with other substances,polyethylene glycol conjugates, i.e. so-called pegylated form of thepolypeptide.

As used herein “a human granulocyte-colony stimulating factor” means apolypeptide which has the same or substantially the same amino acidsequence and biological activity as a naturally occurring humangranulocyte-colony stimulating factor. The term “humangranulocyte-colony stimulating factor” thus encompasses polypeptideswhich have one or more additional amino acids, typically less than 5additional amino acids at either the N-terminus or the C-terminus orboth so long as the biological activity is retained, and specificallyincludes a polypeptide having an N-terminal methionine added to thesequence present in a naturally occurring granulocyte-colony stimulatingfactor. The term also encompasses conjugates with other substances,polyethylene glycol conjugates, i.e. so-called pegylated form of thepolypeptide.

As used herein “a human granulocyte macrophage-colony stimulatingfactor” means a polypeptide which has the same or substantially the sameamino acid sequence and biological activity as a naturally occurringhuman granulocyte macrophage-colony stimulating factor. The term “humangranulocyte macrophage-colony stimulating factor” thus encompassespolypeptides which have one or more additional amino acids, typicallyless than 5 additional amino acids at either the N-terminus or theC-terminus or both so long as the biological activity is retained, andspecifically includes a polypeptide having an N-terminal methionineadded to the sequence present in a naturally occurring granulocytemacrophage-colony stimulating factor. The term also encompassesconjugates with other substances, polyethylene glycol conjugates, i.e.so-called pegylated form of the polypeptide.

As used herein “a human interleukin 8” means a polypeptide which has thesame or substantially the same amino acid sequence and biologicalactivity as a naturally occurring human interleukin 8. The term “humaninterleukin 8” thus encompasses polypeptides which have one or moreadditional amino acids, typically less than 5 additional amino acids ateither the N-terminus or the C-terminus or both so long as thebiological activity is retained, and specifically includes a polypeptidehaving an N-terminal methionine added to the sequence present in anaturally occurring interleukin 8. The term also encompasses conjugateswith other substances, polyethylene glycol conjugates, i.e. so-calledpegylated form of the polypeptide.

As used herein “a human vascular endothelial growth factor” means apolypeptide which has the same or substantially the same amino acidsequence and biological activity as a naturally occurring human vascularendothelial growth factor. The term “human vascular endothelial growthfactor” thus encompasses polypeptides which have one or more additionalamino acids, typically less than 5 additional amino acids at either theN-terminus or the C-terminus or both so long as the biological activityis retained, and specifically includes a polypeptide having anN-terminal methionine added to the sequence present in a naturallyoccurring vascular endothelial growth factor. The term also encompassesconjugates with other substances, polyethylene glycol conjugates, i.e.so-called pegylated form of the polypeptide.

As used herein “a human fibroblast growth factor” means a polypeptidewhich has the same or substantially the same amino acid sequence andbiological activity as a naturally occurring human fibroblast growthfactor. The term “human fibroblast growth factor” thus encompassespolypeptides which have one or more additional amino acids, typicallyless than 5 additional amino acids at either the N-terminus or theC-terminus or both so long as the biological activity is retained, andspecifically includes a polypeptide having an N-terminal methionineadded to the sequence present in a naturally occurring fibroblast growthfactor. The term also encompasses conjugates with other substances,polyethylene glycol conjugates, i.e. so-called pegylated form of thepolypeptide.

In a further embodiment the instant method further comprisesadministering an effective amount of a second agent that increases thecardiomyocyte proliferation caused by the human endothelial progenitorcells. Effective amounts of the second agent are amounts sufficient toenhance or accelerate cardiomyocyte proliferation in the presence ofadministered endothelial progenitor cells. In further embodiments theendothelial progenitor cells express CD117, CD34, AC133 or a high levelof intracellular GATA-2 activity. In one embodiment the administeringcomprises injecting directly into the subject's peripheral circulation,heart muscle, left ventricle, right ventricle, coronary artery,cerebro-spinal fluid, neural tissue, ischemic tissue, or post-ischemictissue.

In one embodiment the second agent is an antisense oligonucleotide whichspecifically inhibits translation of Vitamin D3 Up-Regulated Protein-1(VDUP-1) mRNA.

Therapeutically useful targeted inhibition of VDUP-1 protein (SEQ IDNO:2) expression can be achieved through the use of antisenseoligonucleotides. Antisense oligonucleotides are small fragments of DNAand derivatives thereof complementary to a defined sequence on aspecified mRNA. A VDUP-1 antisense oligonucleotide specifically binds totargets on the VDUP-1 mRNA (SEQ ID NO:1) molecule and in doing soinhibits the translation thereof into VDUP-1 protein (SEQ ID NO:2).

Antisense oligonucleotide molecules synthesized with a phosphorothioatebackbone have proven particularly resistant to exonuclease damagecompared to standard deoxyribonucleic acids, and so they are used inpreference. A phosphorothioate antisense oligonucleotide for VDUP-1 mRNAcan be synthesized on an Applied Biosystems (Foster City, Calif.) model380B DNA synthesizer by standard methods. E.g. Sulfurization can beperformed using tetraethylthiuram disulfide/acetonitrile. Followingcleavage from controlled pore glass support, oligodeoxynucleotides canbe base deblocked in ammonium hydroxide at 60° C. for 8 h and purifiedby reversed-phase HPLC [0.1M triethylammonium bicarbonate/acetonitrile;PRP-1 support]. Oligomers can be detritylated in 3% acetic acid andprecipitated with 2% lithiumperchlorate/acetone, dissolved in sterilewater and re-precipitated as the sodium salt from 1 M NaCl/ethanol.Concentrations of the full length species can be determined by UVspectroscopy.

Any other means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives.

Hybridization of antisense oligonucleotides with VDUP-1 mRNA interfereswith one or more of the normal functions of VDUP-1 1 mRNA. The functionsof mRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in by theRNA.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and borano-phosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Also, oligonucleotides havinginverted polarity comprise a single 3′ to 3′ linkage at the 3′-mostinternucleotide linkage i.e. a single inverted nucleoside residue whichmay be abasic (the nucleobase is missing or has a hydroxyl group inplace thereof) are included. Various salts, mixed salts and free acidforms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

In accordance with this invention, persons of ordinary skill in the artwill understand that messenger RNA includes not only the information toencode a protein using the three letter genetic code, but alsoassociated ribonucleotides which form a region known to such persons asthe 5′-untranslated region, the 3′-untranslated region, the 5′ capregion and intron/exon junction ribonucleotides. Thus, antisenseoligonucleotides or the catalytic nucleic acids described below may beformulated in accordance with this invention which are targeted whollyor in part to these associated ribonucleotides as well as to theinformational ribonucleotides. The antisense oligonucleotides maytherefore be specifically hybridizable with a transcription initiationsite region, a translation initiation codon region, a 5′ cap region, anintron/exon junction, coding sequences, a translation termination codonregion or sequences in the 5′- or 3′-untranslated region. Similarly, thecatalytic nucleic acids may specifically cleave a transcriptioninitiation site region, a translation initiation codon region, a 5′ capregion, an intron/exon junction, coding sequences, a translationtermination codon region or sequences in the 5′- or 3′-untranslatedregion. As is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule). A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterm “translation initiation codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine in eukaryotes. It is also known in the art that eukaryoticgenes may have two or more alternative translation initiation codons,any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “translationinitiation codon” refers to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding VDUP-1, regardless of the sequence(s) of such codons. It isalso known in the art that a translation termination codon of a gene mayhave one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively). The term “translation initiation codon region” refers toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. This region is one preferred targetregion. Similarly, the term “translation termination codon region”refers to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation termination codon. This region is also onepreferred target region. The open reading frame or “coding region,”which is known in the art to refer to the region between the translationinitiation codon and the translation termination codon, is also a regionwhich may be targeted effectively. Other preferred target regionsinclude the 5′ untranslated region (5′UTR), known in the art to refer tothe portion of an mRNA in the 5′ direction from the translationinitiation codon, and thus including nucleotides between the 5′ cap siteand the translation initiation codon of an mRNA or correspondingnucleotides on the gene, and the 3′ untranslated region (3′UTR), knownin the art to refer to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. mRNA splice sites may also bepreferred target regions, and are particularly useful in situationswhere aberrant splicing is implicated in disease, or where anoverproduction of a particular mRNA splice product is implicated indisease. Aberrant fusion junctions due to rearrangements or deletionsmay also be preferred targets.

Once the target site or sites have been identified, antisenseoligonucleotides can be chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired disruption of the function of themolecule. “Hybridization”, in the context of this invention, meanshydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases, usually on opposite nucleic acid strands or tworegions of a nucleic acid strand.

Guanine and cytosine are examples of complementary bases which are knownto form three hydrogen bonds between them. Adenine and thymine areexamples of complementary bases which form two hydrogen bonds betweenthem. “Specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity such thatstable and specific binding occurs between the DNA or RNA target and thecatalytic nucleic acids. Similarly, catalytic nucleic acids aresynthesized once cleavage target sites on the VDUP-1 mRNA molecule.

It is preferred to administer antisense oligonucleotides or catalyticnucleic acids or analogs thereof or other agents to mammals sufferingfrom cardiovascular disease, in either native form or suspended in acarrier medium in amounts and upon treatment schedules which areeffective to therapeutically treat the mammals to reduce the detrimentaleffects of cardiovascular disease. One or more different catalyticnucleic acids or antisense oligonucleotides or analogs thereof targetingdifferent sections of the nucleic acid sequence of VDUP-1 mRNA may beadministered together in a single dose or in different doses and atdifferent amounts and times depending upon the desired therapy. Thecatalytic nucleic acids or antisense oligonucleotides can beadministered to mammals in a manner capable of getting theoligonucleotides initially into the blood stream and subsequently intocells, or alternatively in a manner so as to directly introduce thecatalytic nucleic acids or antisense oligonucleotides into the cells orgroups of cells, for example cardiomyocytes, by such means byelectroporation or by direct injection into the heart. Antisenseoligonucleotides whose presence in cells can inhibit transcription orprotein synthesis can be administered by intravenous injection,intravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, orally or rectally. Human pharmacokinetics of certainantisense oligonucleotides have been studied. See (105) incorporated byreference in its entirety. It is within the scale of a person's skill inthe art to determine optimum dosages and treatment schedules for suchtreatment regimens.

Any of the agents employed in this invention can be administeredintracoronary or intramyocardially, for example by injection. The agentscan be delivered in various means as otherwise discussed, including bystents, such as drug-eluting stents, and in time-release formats such asslow-release. Where the agents are proteins, including but not limitedto SDF-1, G-CSF, GM-CSF, and VEGF, the agents can be administereddirectly or indirectly, or may be administered via induction of geneexpression, through gene therapy, adenoviral delivery and other suchmethods familiar to those skilled in the art.

Doses of the oligonucleotides or analogs thereof of the presentinvention in a pharmaceutical dosage unit will be an efficacious,nontoxic quantity administered to a human patient in need ofcardiomyocyte regeneration (or inhibition of VDUP-1 expression) from 1-6or more times daily or every other day. Dosage is dependent on severityand responsiveness of the effects of abnormal cardiovascular disease tobe treated, with course of treatment lasting from several days to monthsor until a cure is effected or a reduction of the effects is achieved.Oral dosage units for human administration generally use lower doses.The actual dosage administered may take into account the size and weightof the patient, whether the nature of the treatment is prophylactic ortherapeutic in nature, the age, weight, health and sex of the patient,the route of administration, and other factors.

In another embodiment the second agent is a pro-angiogenic agent. Infurther embodiments the pro-angiogenic agent is vascular endothelialgrowth factor, fibroblast growth factor or angiopoietin. In anotherembodiment the second agent induces expression of a pro-angiogenicfactor. In a further embodiment the second agent is Hypoxia InducibleFactor-1.

In another embodiment the second agent is a catalytic nucleic acid whichspecifically inhibits translation of Vitamin D3 Up-Regulated Protein-1mRNA. In a further embodiment the catalytic nucleic acid comprisesdeoxyribonucleotides. In another embodiment the catalytic nucleic acidcomprises ribonucleotides.

Catalytic nucleic acid molecules can cleave Vitamin D3 Up-RegulatedProtein-1 (VDUP-1) mRNA (corresponding DNA shown in SEQ ID NO:1, FIG. 5)at each and any of the consensus sequences therein. Since catalyticribo- and deoxyribo-nucleic acid consensus sequences are known, and theVDUP-1 Protein mRNA sequence is known, one of ordinary skill couldreadily construct a catalytic ribo- or deoxyribo nucleic acid moleculedirected to any of the VDUP-1 protein mRNA consensus sequences based onthe instant specification. In preferred embodiments of this inventionthe catalytic deoxyribonucleic acids include the 10-23 structure.Examples of catalytic ribonucleic acids include hairpin and hammerheadribozymes. In preferred embodiments of this invention, the catalyticribonucleic acid molecule is formed in a hammerhead (50) or hairpinmotif (51, 52, 53), but may also be formed in the motif of a hepatitisdelta virus (54), group I intron (60), RNaseP RNA (in association withan RNA guide sequence) (55,56) or Neurospora VS RNA (57, 58, 59).

To target the VDUP-1 mRNA (SEQ ID NO:1), catalytic nucleic acids can bedesigned based on the consensus cleavage sites 5′-purine:pyrimidine-3′in the VDUP-1 mRNA sequence (104) (see FIGS. 6-10) for cleavage sites onDNA corresponding to the mRNA encoding VDUP-1 (SEQ ID NO:2). Thosepotential cleavage sites located on an open loop of the mRNA accordingto RNA folding software e.g. RNADRaw 2.1 are particularly preferred astargets (61). The DNA based catalytic nucleic acids can utilize thestructure where two sequence-specific arms are attached to a catalyticcore based on the VDUP-1 mRNA sequence. Further examples of catalyticDNA structure are detailed in (62) and (63). Commercially availablemouse brain polyA-RNA (Ambion) can serve as a template in the in vitrocleavage reaction to test the efficiency of the catalyticdeoxyribonucleic acids. Catalytic RNA, is described above, is designedsimilarly. Hammerhead ribozymes can cleave any 5′-NUH-3′ triplets of amRNA, where U is conserved and N is any nucleotide and H can be C,U,A,but not G. For example, the sites which can be cleaved by a hammerheadribozyme in human VDUP-1 mRNA coding region are shown in FIG. 10.

Cleaving of VDUP-1 mRNA with catalytic nucleic acids interferes with oneor more of the normal functions of VDUP-1 mRNA. The functions of mRNA tobe interfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.

The nucleotides may comprise other bases such as inosine, deoxyinosine,hypoxanthine may be used. In addition, isoteric purine2′deoxy-furanoside analogs, 2′-deoxynebularine or 2′deoxyxanthosine, orother purine or pyrimidine analogs may also be used. By carefullyselecting the bases and base analogs, one may fine tune thehybridization properties of the oligonucleotide. For example, inosinemay be used to reduce hybridization specificity, while diaminopurinesmay be used to increase hybridization specificity.

Adenine and guanine may be modified at positions N3, N7, N9, C2, C4, C5,C6, or C8 and still maintain their hydrogen bonding abilities. Cytosine,thymine and uracil may be modified at positions N1, C2, C4, C5, or C6and still maintain their hydrogen bonding abilities. Some base analogshave different hydrogen bonding attributes than the naturally occurringbases. For example, 2-amino-2′-dA forms three (3), instead of the usualtwo (2), hydrogen bonds to thymine (T). Examples of base analogs thathave been shown to increase duplex stability include, but are notlimited to, 5-fluoro-2′-dU, 5-bromo-2′-dU, 5-methyl-2′-dC,5-propynyl-2′-dC, 5-propynyl-2′-dU, 2-amino-2′-dA, 7-deazaguanosine,7-deazadenosine, and N2-Imidazoylpropyl-2′-dG.

Nucleotide analogs may be created by modifying and/or replacing a sugarmoiety. The sugar moieties of the nucleotides may also be modified bythe addition of one or more substituents. For example, one or more ofthe sugar moieties may contain one or more of the followingsubstituents: amino, alkylamino, araalkyl, heteroalkyl,heterocycloalkyl, aminoalkylamino, O, H, an alkyl, polyalkylamino,substituted silyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl,SOMe, SO₂Me, ONO₂, NH-alkyl, OCH₂CH═CH₂, OCH₂CCH, OCCHO, allyl, O-allyl,NO₂, N₃, and NH₂. For example, the 2′ position of the sugar may bemodified to contain one of the following groups: H, OH, OCN, O-alkyl, F,CN, CF₃, allyl, O-allyl, OCF₃, S-alkyl, SOMe, SO₂Me, ONO, NO₂, N₃, NH₂,NH-alkyl, or OCH═CH₂, OCCH, wherein the alkyl may be straight, branched,saturated, or unsaturated. In addition, the nucleotide may have one ormore of its sugars modified and/or replaced so as to be a ribose orhexose (i.e. glucose, galactose) or have one or more anomeric sugars.The nucleotide may also have one or more L-sugars.

Representative United States patents that teach the preparation of suchmodified bases/nucleosides/nucleotides include, but are not limited to,U.S. Pat. Nos. 6,248,878, and 6,251,666 which are herein incorporated byreference.

The sugar may be modified to contain one or more linkers for attachmentto other chemicals such as fluorescent labels. In an embodiment, thesugar is linked to one or more aminoalkyloxy linkers. In anotherembodiment, the sugar contains one or more alkylamino linkers.Aminoalkyloxy and alkylamino linkers may be attached to biotin, cholicacid, fluorescein, or other chemical moieties through their amino group.

Nucleotide analogs or derivatives may have pendant groups attached.Pendant groups serve a variety of purposes which include, but are notlimited to, increasing cellular uptake of the oligonucleotide, enhancingdegradation of the target nucleic acid, and increasing hybridizationaffinity. Pendant groups can be linked to any portion of theoligonucleotide but are commonly linked to the end(s) of theoligonucleotide chain. Examples of pendant groups include, but are notlimited to: acridine derivatives (i.e.2-methoxy-6-chloro-9-aminoacridine); cross-linkers such as psoralenderivatives, azidophenacyl, proflavin, and azidoproflavin; artificialendonucleases; metal complexes such as EDTA-Fe(II),o-phenanthroline-Cu(I), and porphyrin-Fe(II); alkylating moieties;nucleases such as amino-1-hexanolstaphylococcal nuclease and alkalinephosphatase; terminal transferases; abzymes; cholesteryl moieties;lipophilic carriers; peptide conjugates; long chain alcohols; phosphateesters; amino; mercapto groups; radioactive markers; nonradioactivemarkers such as dyes; and polylysine or other polyamines. In oneexample, the nucleic acid comprises an oligonucleotide conjugated to acarbohydrate, sulfated carbohydrate, or gylcan. Conjugates may beregarded as a way as to introduce a specificity into otherwiseunspecific DNA binding molecules by covalently linking them to aselectively hybridizing oligonucleotide.

The catalytic nucleic acid binding domains (i.e. the non-catalyticdomains) or antisense oligonucleotide may comprise modified bonds. Forexample internucleosides bonds of the oligonucleotide may comprisephosphorothioate linkages. The nucleic acid may comprise nucleotideshaving moiety may be modified by replacing one or both of the twobridging oxygen atoms of the linkage with analogues such as —NH, —CH, or—S. Other oxygen analogues known in the art may also be used. Thephosphorothioate bonds may be stereo regular or stereo random.

The oligonucleotide moiety may have one or more of its sugars modifiedor replaced so as to be ribose, glucose, sucrose, or galactose, or anyother sugar. Alternatively, the phosphorothioate oligonucleotide mayhave one or more of its sugars substituted or modified in its 2′position, i.e. 2′allyl or 2′-O-allyl. An example of a 2′-O-allyl sugaris a 2′-O-methylribonucleotide. Further, the phosphorothioateoligonucleotide may have one or more of its sugars substituted ormodified to form an α-anomeric sugar.

A catalytic nucleic acid may include non-nucleotide substitution. Thenon-nucleotide substitution includes either abasic nucleotide,polyether, polyamine, polyamide, peptide, carbohydrate, lipid orpolyhydrocarbon compounds. The term “abasic” or “abasic nucleotide” asused herein encompasses sugar moieties lacking a base or having otherchemical groups in place of base at the 1′ position.

Determining the effective amount of the instant nucleic acid moleculescan be done based on animal data using routine computational methods. Inone embodiment, the effective amount contains between about 10 ng andabout 100 μg of the instant nucleic acid molecules per kg body mass. Inanother embodiment, the effective amount contains between about 100 ngand about 10 μg of the nucleic acid molecules per kg body mass. In afurther embodiment, the effective amount contains between about 1 μg andabout 5 μg, and in a further embodiment about 2 μg, of the nucleic acidmolecules per kg body mass.

In an embodiment the second agent promotes trafficking and is a CCchemokine. In further embodiments the CC chemokine is RANTES, EOTAXIN,monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, or MCP.

In an embodiment the second agent promotes trafficking and is a CXCchemokine. In further embodiments the CXC chemokine is Interleukin-8,Gro-Alpha, or Stromal-Derived Factor-1.

In an embodiment the second agent promotes mobilization of angioblastsinto the subject's bloodstream.

In an embodiment the cardiac progenitor cell is resident in the heart,or that comes into the heart from elsewhere after acute ischemia, issmaller than mature cardiomyocytes, expresses alpha sarcomeric actin butis negative for troponin, is normally quiescent but can be induced to gointo cell cycle as defined by positive Ki67 staining.

This invention further provides a method of improving cardiac functionin a subject comprising administering to the subject an amount of anagent effective to induce proliferation of cardiomyocytes in the hearttissue in the subject so as to thereby improve cardiac function in thesubject.

Cardiac function, or myocardial function, can be determined by anycombination of improvement in ejection fraction, perfusion, reduction inanginal symptoms, improvement in quality of life, increased walkingability, longevity of life, reduction in heart medication, and/orprevention of heart failure, as this terms are normally understood inthe art. An improvement in cardiac function in a subject is measured bydetermining any of these factors in a subject before and after treatmentby any of the instant methods. In one embodiment of the instant methods,the subject has suffered myocardial ischemia or myocardial infarct. Indifferent embodiments the agent is G-CSF, GM-CSF, IL-8, a Gro familychemokine, an inhibitor of CXCR4, or an inhibitor of SDF-1. In furtherembodiments the inhibitor of CXCR4 is a small molecule or a monoclonalantibody, the inhibitor of CXCR4 is a small molecule and is AMD 3100,the inhibitor of SDF-1 is a small molecule or a monoclonal antibody, andthe Gro family chemokine is Gro alpha. In another embodiment theinhibitor is AMD 070. In differing embodiments the the inhibitor ofCXCR4 is a catalytic nucleic acid, an oligonucleotide, RNAi, (RNAinterference) or a small molecule.

This invention further provides a method of inducing apoptosis in a cellcomprising inhibiting expression of a peroxiredoxin in the cell. In oneembodiment the cell is a tumor cell. In different embodiments theperoxiredoxin is peroxiredoxin I, II, II, IV, or V. In one embodimentthe inhibition of expression of peroxiredoxin is effected by contactingthe cell with a catalytic nucleic acid which binds mRNA encodingperoxiredoxin, thereby inhibiting the expression thereof. In differentembodiments the inhibition of expression of peroxiredoxin is effected bycontacting the cell with an antisense oligonucleotide, a monoclonalantibody, RNA interference or a small molecule.

This invention further provides a method of treating a disorder of atissue of a subject involving loss of tissue cells which comprisesadministering to the subject an amount of an agent effective to causetissue cell proliferation within the tissue of the subject so as tothereby treat the disorder. In differing embodiments the tissue iscardiac tissue, brain tissue, peripheral vascular tissue, hepatictissue, renal tissue, gastrointestinal tissue, lung tissue, smoothmuscle tissue, or striated muscle tissue. In differing embodiments theagent is G-CSF, SDF-1, GM-CSF, IL-8, VEGF. In one embodiment the agentis an inhibitor of CXCR4/SDF-1 interaction. In further embodiments theinhibitor is a catalytic nucleic acid, a monoclonal antibody, aantisense oligonucleotide, a small molecule, or RNAi. In one embodimentthe agent is an inducer of peroxiredoxin expression. In furtherembodiments the peroxiredoxin is peroxiredoxin I, II, III, IV, or V.

In any of the instant methods wherein the agent is G-CSF, the G-CSF maybe a covalent conjugate of recombinant methionyl human G-CSF(Filgrastim) and monomethoxypolyethylene glycol, such as Neulasta.Filgrastim is a water-soluble 175 amino acid protein with a molecularweight of approximately 19 kilodaltons (kd). Filgrastim is obtained fromthe bacterial fermentation of a strain of Escherichia coli transformedwith a genetically engineered plasmid containing the human G-CSF gene.To produce pegfilgrastim, a 20 kd monomethoxypolyethylene glycolmolecule is covalently bound to the N-terminal methionyl residue ofFilgrastim. The average molecular weight of pegfilgrastim isapproximately 39 kd. Neulasta is supplied in 0.6 mL prefilled syringesfor subcutaneous (SC) injection. Each syringe contains 6 mgpegfilgrastim (based on protein weight), in a sterile, clear, colorless,preservative-free solution (pH 4.0) containing acetate (0.35 mg),sorbitol (30.0 mg), polysorbate 20 (0.02 mg), and sodium (0.02 mg) inwater for injection, USP.

This invention provides a method of treating a disorder of a tissue of asubject involving apoptosis of cells in the tissue which comprisesadministering to the subject an amount of an agent effective to inhibitapoptosis of cells in the tissue within the subject so as to therebytreat the disorder. In one embodiment the agent is an inhibitor ofVDUP-1 expression. In further embodiments the inhibitor of VDUP-1expression is catalytic nucleic acid, a monoclonal antibody, anantisense oligonucleotide, a small molecule, or an RNAi. In differingembodiments the tissue is cardiac tissue, cerebrovascular tissue, orcerebral tissue.

This invention also provides a method of inhibiting proliferation offibroblasts or inflammatory cells in a tissue and thereby inhibitingcollagen formation comprising contacting the tissue with an inhibitor ofVDUP-1 expression. In one embodiment the inhibitor of VDUP-1 expressionis a catalytic nucleic acid, a monoclonal antibody, an antisenseoligonucleotide, a small molecule, or an RNAi.

In one embodiment the subject of any of the above methods is a mammaland in a preferred further embodiment the mammal is a human being. Inone embodiment the subject has a cardiovascular disease. In furtherembodiments the subject has congestive heart failure, has suffered amyocardial infarct, has suffered myocardial ischemia, has angina, or hasa cardiomyopathy.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Results (I) CXC Chemokines Regulate Endothelial ProgenitorCell Migration to the Heart.

We investigated the in vivo role of CXC receptor-ligand interactions inmediating chemotaxis of human Endothelial progenitor cell to ischemictissue and subsequent induction of vasculogenesis, using myocardialinfarction in the LAD-ligated nude rat model. As shown in FIG. 1 a,DiI-labelled human CD34+ cells obtained by G-CSF mobilization (>98% CD34purity, containing 6-12% CD117^(bright) endothelial progenitor cells)were selectively detected in infarcted myocardium after intravenousinjection, but not in myocardium from sham-operated rats.Co-administration of blocking mAbs against rat Cinc (the rat homologueof human IL-8 and Gro-alpha), or against the human surface receptors forthese pro-angiogenic chemokines, CXCR1 or CXCR2, reduced myocardialtrafficking of human bone marrow-derived CD34+ cells at 48 hours by40-60% relative to control antibodies (p<0.01), FIG. 1 b. By two weeks,rats receiving human CD34+ cells demonstrated significantly increasedinfarct bed microvascularity in comparison to rats receiving saline,FIG. 1 c, and this was reduced by 50% when anti-CXCR1/2 mAbs wereco-administered. Since we have previously shown that the vasculogenicproperties of CD34+ cells are abolished after depletion of the minorCD117^(bright) angioblast fraction (7), these results indicate that byregulating angioblast migration to ischemic tissues CXC chemokinesinfluence the development of vasculogenesis at these sites. In contrast,although direct intracardiac injection of IL-8 or SDF-1 at 1.0 μg/mlinto non-infarcted hearts resulted in 2.3 and 2.5-fold increases inmyocardial infiltration by human CD34+ cells at 48 hours (both p<0.01),FIG. 1 d, no vasculogenesis was observed at two weeks under theseconditions. Together, these results indicate that followingchemokine-induced migration to the infarct zone, differentiation ofendothelial progenitor cell to mature endothelial cells and induction ofvasculogenesis require additional factors, as yet undefined, which areproduced under ischemic conditions.

Inhibiting CXCR4/SDF-1 Interactions Redirects Endothelial ProgenitorCells to the Heart.

Although LAD coronary artery ligation resulted in trafficking ofintravenously injected human endothelial progenitor cells to the site ofischemic myocardium, it was also accompanied by increased distributionof human cells to rat bone marrow. As shown in FIG. 2 a, at 2-14 daysafter intravenous injection of 2×10⁶ human CD34+ cells bone marrow fromLAD-ligated rats contained 5-8 fold higher levels of humanCD117^(bright) endothelial progenitor cells compared with bone marrowfrom normal rats, p<0.001. This was presumably due to the proliferativeeffects of factors in ischemic serum since we have previously shown thatculture for 2 days with ischemic serum increases proliferation ofCD34+CD117^(bright) human endothelial progenitor cells by 4-5 fold (7).Because the migration of actively cycling CD34+ cells to bone marrow ispromoted by SDF-1 produced constitutively by marrow stromal cells (31),we investigated whether the distribution of human CD34+CD117^(bright)endothelial progenitor cells to ischemic rat bone marrow involvedSDF-1/CXCR4 interactions. As shown in FIG. 2 b, co-administration ofmAbs against either human CXCR4 or rat SDF-1 significantly inhibitedmigration of intravenously administered human endothelial progenitorcells to ischemic rat bone marrow compared with anti-CD34 controlantibody (both p<0.001). Moreover, co-administration of mAbs againsteither human CXCR4 or rat SDF-1 increased trafficking of CD34+ humanendothelial progenitor cells to ischemic rat myocardium by means of 24%and 17%, respectively (both p<0.001), FIG. 2 c.

Capillary Lumen Size is Dependent on Absolute Angioblast Numbers.

We next examined the relationship between angioblast number, myocardialneovascularization, and protection against myocyte apoptosis. Two daysfollowing LAD ligation animals were intravenously injected with G-CSFmobilized CD34+ human cells reconstituted with varying proportions ofCD117^(bright) endothelial progenitor cells (10³, 10⁵, 10⁵ plusanti-CXCR4 mAb, 2×10⁵, and 2×10⁵ plus anti-CXCR4 mAb). Similar numbersof DiI-labelled human cells were detected in the infarct zone 48 hoursafter injecting each cellular population, data not shown. Induction ofneovascularization at two weeks was measured by performing quantitativeanalysis of medium- and large-sized capillaries, defined, respectively,as having 3-6 or >6 contiguous endothelial lining cells. Medium-sizedcapillaries had mean lumen diameter of 0.020 mm+0.002, while large-sizedcapillaries had mean lumen diameter of 0.053 mm+0.004 (p<0.001).Notably, large-lumen capillaries overlapped in size with arterioleswhich could be distinguished by a thin layer containing 2-3 smoothmuscle cells of rat origin, as determined by positive staining withdesmin and rat MHC class I mAbs. As shown in FIGS. 3 a-c, both the groupreceiving 2×10⁵ endothelial progenitor cells and the one receiving 10⁵endothelial progenitor cells plus anti-CXCR4 mAb demonstrated 1.7-foldhigher numbers of medium-sized capillaries compared with, the other twogroups (p<0.01). The group receiving 2×10⁵ endothelial progenitor cellsadditionally demonstrated 3.3-fold higher numbers of large-lumencapillaries compared with the groups receiving 10³ or 10⁵ endothelialprogenitor cells (p<0.01), and 2-fold higher numbers of large-lumencapillaries compared with the group receiving 10⁵ endothelial progenitorcells plus anti-CXCR4 mAb (p<0.01). As shown in FIG. 3 d,co-administration of anti-CXCR4 mAb together with the highestconcentration of endothelial progenitor cells, 2×10⁵, resulted in afurther 23% increase in growth of large-lumen capillaries. Morestrikingly, there was a further 2-fold increase in capillary numberswhen 2×10⁵ endothelial progenitor cells were injected intravenouslyafter direct intracardiac delivery of 1.0 μg/ml SDF-1 into infarctedhearts (p<0.01). Since no similar increase in peri-infarct capillarynumbers was seen after intracardiac delivery of IL-8, we interpret theseresults to indicate that the endogenous IL-8 concentrations in ischemicrat hearts were sufficient to saturate angioblast CXCR1/2 receptors invivo, whereas intracardiac injection of SDF-1 resulted in a shift in thebalance of SDF-1 expression between bone marrow and heart, andconsequently resulted in redirected angioblast trafficking to theischemic heart. As shown in FIG. 3 e, the number of apoptoticcardiomyocytes at the infarct rim was significantly reduced in both ratsreceiving 10⁵ endothelial progenitor cells plus anti-CXCR4 mAb and thosereceiving 2×10⁵ endothelial progenitor cells compared with the groupsreceiving either 10³ or 10⁵ endothelial progenitor cells alone (bothp<0.001). Moreover, co-administration of anti-CXCR4 mAb or intracardiacinjection of SDF-1 resulted in further reductions in cardiomyocyteapoptosis of 65% and 76%, respectively, FIG. 3 f (both p<0.001).Together, these data indicate that post-infarct cardioprotection againstmyocyte apoptosis is dependent on myocardial neovascularization inducedby a critical number of intravenously injected endothelial progenitorcells. This threshold can apparently be lowered by strategies thatprevent endothelial progenitor cell redistribution to the bone marrow,such as interrupting CXCR4/SDF-1 interactions, or enhanced SDF-1expression in the ischemic myocardium.

Capillary Lumen Size as Determinant of Improvement in Cardiac Function.

We next examined the effect of increasing the number of humanendothelial progenitor cells trafficking to ischemic myocardium onlong-term myocardial function, defined as the degree of improvement inleft ventricular ejection fraction (LVEF) and reduction in leftventricular end-systolic area (LVAs) at 15 weeks after intravenousinjection, FIGS. 4 a and b. No improvement in these parameters wasobserved in the groups receiving 10³ or 10⁵ endothelial progenitor cellsin comparison to rats receiving saline alone. In contrast, ratsreceiving 10⁵ endothelial progenitor cells plus anti-CXCR4 mAbdemonstrated significant improvement in these parameters, 22+2% meanrecovery in LVEF and 24+4% mean reduction in LVAs (both p<0.001). Evenmore strikingly, the group receiving 2×10′ endothelial progenitor cellshad a mean recovery in LVEF of 34+4% and a mean reduction in LVAs of37+6% (both p<0.001), or 50% further improvement in both parameters.These results were very surprising to us since both groups of animalshad demonstrated the same degree of neovascularization involvingmedium-sized capillaries at two weeks together with similar levels ofprotection against early apoptosis of cardiomyocytes. This suggestedthat the additional functional long-term improvement in rats receiving2×10⁵ human endothelial progenitor cells was related to the earlydevelopment of large-sized capillaries and was mediated through adifferent mechanism than protection against myocyte apoptosis.

Large Capillaries Induce Sustained Regeneration of Endogenous Myocytes.

Although myocyte hypertrophy and increase in nuclear ploidy havegenerally been considered the primary mammalian cardiac responses toischemia, damage, and overload (1,2) recent observations have suggestedthat human cardiomyocytes have the capacity to proliferate andregenerate in response to injury (18,19). Therefore, we investigatedwhether the additive improvement in cardiac function observed afterinjection of 2×10⁵ human endothelial progenitor cells involved inductionof cardiomyocyte proliferation and/or regeneration. At two weeks afterLAD ligation rats receiving 2×10⁵ human endothelial progenitor cellsdemonstrated numerous “fingers” of cardiomyocytes of rat origin, asdetermined by expression of rat MHC class I molecules, extending fromthe peri-infarct region into the infarct zone. Similar extensions wereseen less frequently in animals receiving 10³ and 10⁵ endothelialprogenitor cells, and very rarely in those receiving saline. As shown inFIG. 4 c, the islands of cardiomyocytes at the peri-infarct rim inanimals receiving 2×10⁵ human endothelial progenitor cells contained ahigh frequency of rat myocytes with DNA activity, as determined by dualstaining with mAbs reactive against cardiomyocyte-specific troponin Iand rat Ki-67. In contrast, in animals receiving saline there was a highfrequency of cells with fibroblast morphology and reactivity with ratKi-67, but not troponin I, within the infarct zone. The number ofcardiomyocytes progressing through cell cycle at the peri-infarct regionof rats receiving 2×10⁵ human endothelial progenitor cells was 40-foldhigher than that at sites distal to the infarct, where myocyte DNAactivity was no different than in sham-operated rats. As shown in FIG. 4d, animals receiving 2×10⁵ human endothelial progenitor cells had a20-fold higher number of cell-cycling cardiomyocytes at the peri-infarctrim than that found in non-infarcted hearts (1.19+0.2% vs 0.06+0.03%,p<0.01) and 3.5-fold higher than in the same region in LAD-ligatedcontrols receiving saline (1.19+0.2% vs 0.344+0.1%, p<0.01). When 2×10⁵human endothelial progenitor cells were intravenously injected afterdirect intracardiac delivery of 1.0 μg/ml SDF-1 into infarcted hearts,the number of cell-cycling cardiomyocytes at the peri-infarct rim wasincreased by a further 1.9-fold compared with intravenous injection of2×10⁵ human endothelial progenitor cells alone (FIG. 4 e, p<0.01). Thus,intracardiac injection of SDF-1 in combination with intravenousinjection of 2×10⁵ human endothelial progenitor cells resulted inapproximately an 8-fold cumulative increase in cell-cyclingcardiomyocytes at two weeks compared with LAD-ligated controls receivingsaline, and translated into over 4-fold greater LVEF improvement,determined by echocardiography, compared with intravenous injection of2×10⁵ endothelial progenitor cells alone (FIG. 4 f, p<0.01).Co-administration of anti-CXCR4 mAb augmented LVEF improvement by2.8-fold (p<0.01) while intracardiac injection of IL-8 conferred noadditive benefit.

Quantitation of the ratio of fibrous tissue to myocytes at 15 weeksdemonstrated significantly reduced proportions of scar/normal leftventricular myocardium in both the group receiving 2×10⁵ endothelialprogenitor cells and the one receiving 10⁵ endothelial progenitor cellsplus anti-CXCR4 mAb, respectively 13% and 21% compared with 37-46% foreach of the other groups (p<0.01), FIG. 4 g. Since both groups hadnearly identical levels of protection against early cardiomyocyteapoptosis, we infer that the additional 38% reduction in scar/myocyteratio seen in the group injected with 2×10⁵ endothelial progenitor cellsactually reflects proliferation/regeneration of endogenous ratcardiomyocytes induced by nutrient supply from large-vesselneovascularization. This presumably accounts for the increase infunctional improvement seen in this group. We conclude that the ratio ofscar size to left ventricular muscle mass reflects, in part, positiveeffects imparted by the ability of the residual myocardium toproliferate and regenerate, in addition to negative effects of theinitial infarct size and positive effects of anti-apoptotic,cardioprotective mechanisms. The overall effects of medium- andlarge-size neovasculature combining to both protect against myocyteapoptosis and induce myocyte proliferation/regeneration are showndramatically in FIG. 4 h where, in contrast to saline controls,injection with 2×10⁵ endothelial progenitor cells resulted in almostcomplete salvage of the anterior myocardium, normal septal size andminimal collagen deposition.

Effecting Regeneration PAI-1-Inhibiting Catalytic Nucleic Acid AugmentsHuman Angioblast-Dependent Cardiomyocyte Regeneration.

We investigated whether possible neovascularization induced by acatalytic nucleic acid (designated E2) capable of inhibiting expressionof PAI-1 was associated with cardiomyocyte regeneration. Injection of E2alone did not induce cardiomyocyte regeneration despite the increase inneovascularization. Combining E2 injection with intravenously deliveredhuman endothelial progenitor cells strikingly increased the degree ofcardiomyocyte regeneration, to levels 7.5-fold higher than in salinecontrols (p<0.01). A scrambled DNA control enzyme (E0) had no sucheffect. Moreover, whereas E2 alone did not improve myocardial function,as determined by recovery in left ventricular ejection fraction at twoweeks combining E2 with human endothelial progenitor cells resulted inalmost doubling of the positive effect of endothelial progenitor cellsalone on cardiac functional recovery. These results emphasize theimportance of cardiomyocyte regeneration as the primary mechanism bywhich cardiac function is improved after infarction. Since combining E2with human endothelial progenitor cells resulted in 62% greater numbersof large capillaries at the peri-infarct, rim than use of eitherapproach alone, these results indicate that angioblast-inducedcardiomyocyte regeneration and improvement in cardiac function can beoptimized by use of synergistic approaches, such as strategies thatinhibit PAI-1 expression, which augment neovascularization eitherdirectly or through angioblast-dependent processes.

We further discovered that intravenous injection of humanCD34+CD117bright angioblasts resulted in a four-fold increase inproliferation/regeneration of rat cardiomyocytes at the peri-infarctregion relative to saline controls, as determined by dual staining fortroponin and Ki67 (p<0.01). Combining E2 injection with intravenouslydelivered human angioblasts strikingly increased the degree ofcardiomyocyte regeneration, to levels 7.5-fold higher than in salinecontrols (p<0.01). The scrambled DNA enzyme E0 had no such effect.

Gene Effects on Myocyte Proliferation

We hypothesized that myocardial neovascularization induces the signalsrequired to elicit myocyte proliferation, and therapeutic interventionmimicking this could have striking implications for repair andregeneration of hearts damaged by episodes of ischemia or other insults.

To begin approaching this complex problem, we have employed thetechnique of cDNA Subtractive Hybridization. This technique enablescomparison of the pattern of gene expression between two different setsof conditions. Our initial approach was to compare which genes aredifferentially expressed between hearts from normal rats and rats whohave undergone left anterior descending (LAD) coronary artery ligation48 hours earlier. We hypothesized that whatever the altered pattern ofgene over- or underexpression after 48 hours of ischemia,neovascularization would result in a reversal in the pattern towardsthat seen in the non-ischemic rat heart.

Using cDNA subtractive hybridization we observed a striking reciprocalchange in expression of a group of genes whose function is linkedthrough their regulation of cellular apoptosis and cell cycleprogression following oxidative stress and other inducers of DNA damage.Whereas expression of certain antioxidant genes such as superoxidedismutase was upregulated in ischemic tissue, the antioxidant stressresponsive genes induced by hemin, notably heme binding protein 23(HBP23) and glutathione-S-transferase, were downregulated. Moreover, arecently-identified protein, Vitamin D3 Up-Regulated Protein1(VDUP1),whose mRNA expression is induced by hydrogen peroxide (H₂O₂) followingoxidative stress and whose function counterbalances that of HBP23, wasupregulated in ischemic hearts. These findings are particularly strikingwhen considered in the context of previous observations that thepresence of deficits in antioxidants and increased oxidative stressaccompanying myocardial infarction appear to be directly implicated inthe pathogenesis of post-infarct heart failure (74,75).

As shown in FIG. 5, using RT-PCR, the reciprocal changes in mRNAexpression of HBP23 and VDUP1 in ischemic vs normal rat hearts wereconfirmed. Moreover, mRNA expression of these two genes returned tonormal in rat tissue two weeks after intravenous injection of humanadult bone marrow-derived progenitors and infarct zoneneovascularization. In contrast, in LAD-ligated rat hearts receivingsaline, no change in mRNA expression of these genes was observed at twoweeks in comparison to the pattern observed at 48 hours. To comprehendthe relationship between the effects of neovascularization on thesealtered expression patterns of HBP23 and VDUP1 following ischemia, andthe observed protection against cardiomyocyte apoptosis together withinduction of cardiomyocyte proliferation/regeneration, it is importantto understand in detail the molecular effects of the products encoded bythese genes on cellular apoptosis and cell cycle progression.

When cells proliferate, the mitotic cycle progression is tightlyregulated by an intricate network of positive and negative signals.Progress from one phase of the cell cycle to the next is controlled bythe transduction of mitogenic signals to cyclically expressed proteinsknown as cyclins and subsequent activation or inactivation of severalmembers of a conserved family of serine/threonine protein kinases knownas the cyclin-dependent kinases (cdks) (67). Growth arrest observed withsuch diverse processes as DNA damage, terminal differentiation, andreplicative senescence is due to negative regulation of cell cycleprogression by two functionally distinct families of Cdk inhibitors, theInk4 and Cip/Kip families (64). The cell cycle inhibitory activity ofp21Cip1/WAF1 is intimately correlated with its nuclear localization andparticipation in quaternary complexes of cell cycle regulators bybinding to G1 cyclin-CDK through its N-terminal domain and toproliferating cell nuclear antigen (PCNA) through its C-terminal domain(68-71). The latter interaction blocks the ability of PCNA to activateDNA polymerase, the principal replicative DNA polymerase (72). For agrowth-arrested cell to subsequently enter an apoptotic pathway requiressignals provided by specific apoptotic stimuli in concert withcell-cycle regulators. For example, caspase-mediated cleavage of p21,together with upregulation of cyclin A-associated cdk2 activity, havebeen shown to be critical steps for induction of cellular apoptosis byeither deprivation of growth factors (73) or hypoxia of cardiomyocytes(74).

The apoptosis signal-regulating kinase 1 (ASK1) is a pivotal componentin the mechanism of cytokine- and stress-induced apoptosis (75,76).Under basal conditions, resistance to ASK1-mediated apoptosis appears tobe the result of complex formation between ASK1, cytoplasmicp21Cip1/WAF1 (77), and the thiol reductase thioredoxin (TRX) (78).Intact cytoplasmic expression of p21Cip1/WAF1 appears to be importantfor both prevention of apoptosis in response to ASK1 (75) and inmaintaining a state of terminal differentiation (77). Moreover, thereduced form of TRX, but not the oxidized form, binds to the N-terminalportion of ASK1 and is a physiologic inhibitor of ASK1-mediated cellularapoptosis (78). The recently-identified protein VDUP1 has been shown tocompete with ASK1 for binding of the reduced form of TRX (78,79),resulting in augmention of ASK1-mediated apoptosis (80). This indicatesthat ASK1-mediated cellular apoptosis is increased by processes thatresult in a net dissociation of TRX from ASK1, such as either generationof TRX-VDUP1 complexes or generation of oxidised TRX by changes incellular redox status accompanying oxidative stress.

TRX and glutathione constitute the major cellular reducing systems thatmaintain the thiol-disulfide status of the cytosol (81). Theredox-active/dithiol active site of TRX is highly conserved across allspecies, Trp-Cys-Gly-Pro-Cys-Lys. The two cysteine residues at theactive site, Cys-32 and Cys-35, undergo reversible oxidation-reductionreactions catalyzed by a NADPH-dependent enzyme TRX reductase. Thesereactions involve electron transfer via disulfide bridges formed withmembers of a family of antioxidant enzymes known as peroxiredoxins(Prxs), which show peroxidase activity (82,83). Prxs are distinct fromother peroxidases in that they have no cofactors, such as metals orprosthetic groups. Prxs generally have two conserved cysteines at the N-and C-terminal regions (84), and their antioxidant effects are coupledwith the physiological electron donor activity of the TRX system (82,85, 86). Prxs with 95-97% sequence homology have been identified in rats(Herne-binding protein 23, HBP23) (87), mice (mouse macrophage stressprotein 23, MSP23) (88) and humans (proliferation-associated geneproduct, PAG (89) and human natural killer cell-enhancing factor A(90)).

Prxs are members of a repertoire of oxidative stress responsive geneswhose expression is regulated by NF-E2-related factor 2 (Nrf2) whichbinds to an anti-oxidant responsive element (ARE) present in thepromoter of each (91). These include glutathione-S-transferase, hemeoxygenase-1, and TRX. Under basal conditions, Nrf2 is bound to aspecific protein, Keap1, in the cytosol (92). However, under conditionsof oxidative stress Nrf2 dissociates from Keap1 and translocates to thenucleus where it induces transcriptional activation of the anti-oxidantgenes containing ARE motifs. Although the precise extracellularsignalling pathways have not been elucidated, nuclear translocation ofNrf2 and subsequent ARE activation appear to be dependent on pathwaysactivated by phosphatidylinositol 3-kinase (P13 kinase) (93). Inaddition, hemin is a potent inducer of Nrf2 dissociation from Keap1,resulting in TRX gene transcription through the ARE (94).

During periods of rapid changes in cellular redox Prxs presumably serveto maintain the cytosolic levels of reduced TRX by accepting electronsfrom the oxidized form of TRX. This homeostatic mechanism likely enablesmaintenance of sufficient levels of reduced TRX to ensure adequatebinding to ASK1 and prevention of cellular apoptosis. If the endogenousPrx system is overloaded, as might occur during changes in cellularredox when excess oxidized TRX is generated, cellular apoptosis willoccur through the unopposed effects of ASK1. To counteract this,transcriptional activation of Prxs must occur following oxidative stressvia nuclear translocation of Nrf2. This can be achieved either by Nrf2dissociation from Keap1 via hemin- and PI3 kinase-dependent mechanisms(93,94), or by increasing Nrf2 mRNA and protein expression as occursfollowing increase in oxygen tension (95,96).

In addition to directly interacting with TRX, the Prx gene products(PAG, HBP23, MSP23, NKEF, etc) specifically bind the SH3 domain ofc-Abl, a non-receptor tyrosine kinase, inhibiting its activation byvarious stimuli, including agents that damage DNA (97). c-Abl activationthrough the SH3 domain induces either arrest of the cell cycle in phaseG1 or cellular apoptosis (98). Cell cycle arrest is dependent on thekinase activity of c-Abl (96) and is mediated by the ability of c-Abl todownregulate the activity of the cyclin-dependent kinase Cdk2 and inducethe expression of p21 (99). The apoptotic effects of c-Abl are dependenton the ability of nuclear c-Abl to phosphorylate p73, a member of thep53 family of tumor-suppressor proteins which can induce apoptosis(100,101). Recently, it has been shown that cytoplasmic, rather thannuclear, forms of c-Abl are activated by H₂O₂ and that this results inmitochondrial localization of c-Abl, c-Abl dependent cytochrome crelease, and cellular apoptosis following oxidative stress (102,103). Byassociating with c-Abl in vivo, the PAG gene product (and presumably theother Prxs) can inhibit tyrosine phosphorylation induced by c-Abloverexpression and rescue cells from both the cytostatic andpro-apoptotic effects of the activated c-Abl gene product (97).

Our finding that Nrf2-dependent oxidative stress responsive genes aredownregulated following myocardial ischemia likely reflects directeffects of hemin and oxygen deprivation. The end result of Prxdownregulation in the ischemic heart would be augmentation inASK1-dependent cellular apoptosis as well as Abl-dependent apoptosis andcell cycle arrest. The observed parallel increase in VDUP1 expressionwould further augment ASK1-dependent cellular apoptosis. Thus, the ratioin expression of PAG or other Prx mRNA or protein to VDUP1 mRNA orprotein can form the basis of a diagnostic assay to predict the degreeof risk for cardiomyocyte apoptosis and cell cycle arrest afterischemia, as well as enable monitoring of the response to specifictherapy after myocardial ischemia that protects cardiomyocytes againstapototic death and enhances myocardial proliferation/regeneration.

Reversing the reduced expression of the Prxs following myocardialischemia would increase Prxs in the heart in order to protect theischemic myocardium against apoptosis through both c-Abl inhibition andreduction of oxidised TRX, and to enable cardiomyocyteproliferation/regeneration by inhibiting the effects of c-Abl on cellcycle progression from G1 to S phase.

Increasing Nrf2 mRNA or causing dissociation of Nrf2 protein from Keap1,or preferably cause both to occur simultaneously, in the setting ofmyocardial ischemia in order to increase transcription and activity ofmembers of a repertoire of oxidative stress responsive genes whoseexpression is regulated by binding of Nrf2 to an anti-oxidant responsiveelement (ARE) in their promoters, including the Prxs, TRX andglutathione-S-transferase would result in both protection ofcardiomyocytes against apoptosis as well as induce cardiomyocyte cellcycle progression following oxidative stress.

Reducing the expression of VDUP1 following myocardial ischemia wouldprotect the ischemic myocardium against apoptosis by reducing binding ofTRX to VDUP1, and consequently increasing TRX-ASK1 interactions.

Neovascularization of the myocardium, by either bone marrow-derivedendothelial progenitors or any other process, is an example of onemethod which causes induction of Prx expression and reduction in VDUP1expression after myocardial ischemia, and results in both protectionagainst redox-mediated apoptosis and induction of myocardialproliferation/regeneration.

Small molecules which specifically inhibit binding of Nrf2 to Keap1would be expected to have similar protective effects againstcardiomyocyte apoptosis and to induce myocardialproliferation/regeneration after ischemia. Similarly, small moleculesthat specifically inhibit binding of TRX to VDUP1, would be expected tohave similar protective effects against cardiomyocyte apoptosis afterischemia.

Use of small molecules to specifically inhibit c-Abl tyrosine kinaseactivation after myocardial ischemia would be expected to have similarprotective effects against cardiomyocyte apoptosis and to inducemyocardial proliferation/regeneration after ischemia. A specific exampleof a small molecule to inhibit c-Abl tyrosine kinase activation isSTI-571. Use of this or related molecules after myocardial infarctionwould protect against cardiomyocyte apoptosis and induce myocardialproliferation/regeneration.

DNA Enzyme Specific for VDUP-1 Cleaves Synthetic Rat VDUP-1Oligonucleotide

By subtractive hybridization, we found that mRNA expression of theprotein VDUP1 is significantly increased in the heart after acuteischemia. VDUP1 has been shown to bind the cytosolic proteinthioredoxin, TRX, which functions to maintain the thiol-disulfide statusof the cytosol. By binding to reduced forms of TRX, VDUP1 prevents theability of reduced TRX to undergo reversible oxidation-reductionreactions catalyzed by a NADPH-dependent enzyme, TRX reductase. Thisresults in cellular apoptosis due to excessive cytosolic andmitochondrial production of oxygen radicals.

VDUP1 competes for TRX binding with another cytosolic protein that isnormally bound to reduced TRX, the apoptosis signal-regulating kinase 1(ASK1). ASK-1 is a pivotal component in the mechanism of cytokine- andstress-induced apoptosis. Its activation results in excessivephosphorylation and activation of p38 MAP kinase, a principal mediatorof cellular apoptosis. The reduced form of TRX, but not the oxidisedform, binds to the N-terminal portion of ASK1 and is a physiologicinhibitor of ASK1-mediated cellular apoptosis. Binding of VDUP1 to TRXresults in a net dissociation of ASK1 from TRX, potentially resulting inaugmentation of ASK1-mediated apoptosis via p38 MAP kinase-dependentpathways.

With respect to cardiac overexpression of VDUP1, the anticipated effectswould be those due to excessive p38 MAP kinase activation and oxidativeredox damage, including cardiomyocyte apoptosis, fibroblastproliferation, collagen secretion and scar formation. Similar effectswould be expected to be the consequence of VDUP1 overexpression in othertissues undergoing acute or chronic ischemic injury, for example thebrain following cerebrovascular ischemia/stroke.

We have developed a DNA enzyme targeting the VDUP1 mRNA. The DNA enzyme,once delivered to the ischemic myocardium (or other ischemic tissuessuch as the brain) can inhibit local VDUP1 mRNA and protein expression,thus reducing p38 MAP kinase activation and oxidative damage.

As shown in FIG. 12, at enzyme concentrations ranging from 0.05 uM to 5uM the sequence-specific VDUP1 DNA enzyme cleaved a synthetic rat VDUP1oligonucleotide in a concentration- and time-dependent manner.

VDUP-1 DNAzyme Reduces Fibroblast Proliferation And Protects AgainstCardiomyocyte Apoptosis. As shown in FIG. 13( a), intramyocardialinjection of the rat sequence-specific VDUP1 DNA enzyme at 48 hoursafter LAD ligation resulted in a 75% mean inhibition of proliferatingcardiac fibroblasts in the infarct zone two weeks later in comparison toinjection of scrambled DNA enzyme control (p<0.01). In addition, as seenin FIG. 13( b), injection of VDUP1 DNA enzyme resulted in 20% meanreduction in apoptotic cardiomyocytes at the peri-infarct regionrelative to injection with the scrambled DNA enzyme control (p<0.05).

VDUP-1 DNAzyme Reduces Myocardial Scar And Improves Cardiac FunctionAfter Acute Infarction. Inhibition of fibroblast proliferation andcardiomyocyte apoptosis resulted in significant reduction of mature scardeposition in the infarct zone, from a mean of 35% for animals receivingcontrol scrambled DNA enzyme to a mean of 20% for those receiving VDUP1DNA enzyme, FIG. 14( a) (p<0.01). Most dramatic was the effect oncardiac function. As shown in FIG. 14( b), animals receiving VDUP1 DNAenzyme demonstrated a 50% mean recovery in cardiac function, asdetermined by ejection fraction, whereas no improvement was seen inanimals receiving scrambled control DNA enzyme (p<0.01).

Clearly, VDUP1 DNA enzyme prevents cardiomyocyte apoptosis, cardiacfibroblast proliferation and scar formation, resulting in significantimprovement in cardiac function after acute ischemia. These effects arepresumably due to prevention of p38 MAP kinase activation and protectionagainst redox damage. Similar results might be obtained by administeringthe VDUP1 DNA enzyme to other tissue sites of reduced blood flow such asthe brain after cerebrovascular ischemia.

G-CSF

G-CSF is a more potent inducer of neovascularization after myocardialinfarction than GM-CSF. As shown in FIG. 15( a), rats injectedsubcutaneously with human G-CSF at 10 ug/kg for four days starting attwo days after myocardial infarction induced by left anterior descending(LAD) coronary artery ligation demonstrated approximately 7.5-foldgreater numbers of large-diameter blood vessels at the peri-infarctregion two weeks later relative to saline-treated controls (p<0.01). RatGM-CSF administered at the same dosage regimen was less effective,though still inducing 4-fold greater numbers of large-lumen vessels thanin control animals.

Additionally, G-CSF is a more potent inhibitor of cardiomyocyteapoptosis after myocardial infarction than GM-CSF. G-CSF injection was amore potent agent for protecting against cardiomyocyte apoptosis thanGM-CSF used at the same dosage regimen, FIG. 15( b). G-CSFadministration resulted in 36+16% reduction in the numbers of apoptoticcardiomyocytes at the peri-infarct region by two weeks relative tosaline-treated controls (p<0.01), whereas GM-CSF only reduced apoptoticcardiomyocyte numbers by 12+9%.

G-CSF is a more potent inducer of cardiomyocyte regeneration andfunctional cardiac recovery after myocardial infarction than GM-CSF.Next we examined the effects of bone marrow mobilization oncardiomyocyte cell cycling/regeneration and on functional cardiacrecovery. As shown in FIG. 16( a), rats injected subcutaneously withhuman G-CSF at 10 ug/kg for four days starting at two days aftermyocardial infarction demonstrated 3.2-fold greater numbers of cyclingcardiomyocytes two weeks later at the peri-infarct region relative tosaline-treated controls (p<0.05). Rat GM-CSF administered at the samedosage regimen was less effective, resulting in 2.6-fold greater numbersof cycling cardiomyocytes. As shown in FIG. 16( b), this correlated withfunctional cardiac recovery. Whereas saline-treated animals had a 17%mean loss in cardiac function from day 2 to day 14 after infarction asmeasured by ejection fraction, GM-CSF treated animals had a loss of only10% in cardiac function, and G-CSF treated animals actually had a 10%mean improvement in cardiac function (P<0.01). We interpret thesefunctional data to reflect the superior effects of G-CSF bone marrowmobilization on myocardial neovascularization, protection againstcardiomyocyte apoptosis and induction of cardiomyocyte cell cycling.

Anti-CXCR4 Antibodies

Intravenous administration of anti-CXCR4 antibody increases myocardialneovascularization and improves cardiac function after acute infarction.The major mechanism by which G-CSF causes mobilization of bone marrowelements is through interruption of interactions between the chemokinereceptor CXCR4 on bone marrow resident stem cells and its ligand SDF-1.G-CSF induces both cleavage of the N-terminus of CXCR4 and accumulationof serine proteases which directly cleave and inactivate SDF-1. Toexamine whether similar mechanisms were responsible for the effects ofG-CSF administration on myocardial neovascularization and improvement incardiac function, we investigated the effect of interrupting CXCR4-SDF1interactions by intravenously administering a monoclonal anti-CXCR4antibody 48 hours after LAD ligation. As shown in FIG. 17( a) at twoweeks after antibody administration animals receiving anti-CXCR4 mAbdemonstrated a two-fold increase in neovascularization at theperi-infarct region compared with control animals receiving eithersaline or anti-CXCR2 mAb. Moreover, as seen in FIG. 17( b) anti-CXCR4treated animals demonstrated a mean recovery in ejection fraction of 10%at two weeks whereas those receiving anti-CXCR2 mAb had a mean loss incardiac function of 8% (p<0.05). These data support the concept that theobserved effects of G-CSF administration result from interrupting CXCR4interactions in the bone marrow, enabling endothelial progenitor cellsto be mobilized to the peripheral circulation, and to home to ischemicmyocardium where the resultant neovascularization results in improvementin cardiac function.

SDF-1

SDF-1 mRNA expression is not induced early in acutely ischemicmyocardium, and its late induction is inhibited by GM-CSF. Next, wesought to identify a strategy by which the effects of GM-CSF could beaugmented to approach those seen with G-CSF treatment alone, such asincreasing chemotactic signals in the acutely ischemic myocardium. Sincechemotaxis of CD34+ bone marrow stem cells is regulated by interactionsbetween CXCR4 receptors on the CD34+ cells and the CXC chemokine SDF-1,we investigated whether SDF-1 mRNA expression was induced in the acutelyischemic myocardium. As shown in FIG. 18, no difference in myocardialSDF-1 mRNA was observed at 48 hours after LAD ligation in experimentalanimals relative to non-ischemic controls. By two weeks afterinfarction, myocardial SDF-1 mRNA expression had increased by 3.3-foldcompared with saline-treated controls (p<0.01). We interpreted thisdelayed production of SDF-1 as most likely reflecting elaboration byinfiltrating cells such as macrophages. In contrast, systemic GM-CSFadministration was accompanied by approximately 4.5-fold inhibition inSDF-1 mRNA expression at two weeks, to levels actually lower than innon-ischemic controls. Since SDF-1 is a potent chemotactic factor forendothelial progenitor cells, these data suggested that systemic GM-CSFadministration may result in suboptimal myocardial homing of endothelialprogenitors due to decreased myocardial expression of chemotacticligands such as SDF-1.

Intramyocardial injection of SDF-1 after acute myocardial infarctionresults in neovascularization and protection against cardiomyocyteapoptosis. To determine whether altered SDF-1 expression in the acutelyischemic heart affects endothelial progenitor cell chemotaxis andcardiomyocyte function, we examined the effects of directintramyocardial injection of SDF-1 protein at 48 hours after LADligation. As shown in FIGS. 19 (a) and (b), intramyocardial injection of4 ug/kg human recombinant SDF-1 in a total volume of 0.2 ml at 5peri-infarct sites two days post-LAD ligation resulted by two weeks in a5-fold increase in neovascularization and a 44+9% reduction in apoptoticcardiomyocytes at the peri-infarct region relative to control animalsreceiving intramyocardial saline injections (both p<0.05). The effectsof intramyocardial SDF-1 injection on neovascularization and on extentof protection against cardiomyocyte apoptosis were in strikingly closeparallel to the results obtained with systemic administration of G-CSF.Although addition of subcutaneously administered GM-CSF resulted in asynergistic increase in myocardial neovascularization, no furtherbenefit in protection against cardiomyocyte apoptosis was observed.These results suggest that there is a finite amount of protectionagainst cardiomyocyte apoptosis that can be induced byneovascularization, and that this cannot be improved upon by further byinduction of additional new blood vessels.

Intramyocardial injection of SDF-1 induces cardiomyocyte regeneration.As shown in FIG. 20( a), intramyocardial injection of 4 ug/kg humanrecombinant SDF-1 in a total volume of 0.2 ml at 5 peri-infarct sitestwo days post-LAD ligation resulted in a 4.5-fold increase in the numberof cycling cardiomyocytes at the peri-infarct region relative tosaline-injected controls (p<0.01). Addition of systemic GM-CSFadministration resulted in synergistic effects on cardiomyocyteregeneration. Notably, the numbers of cycling cardiomyocytes at theperi-infarct region in animals receiving combined therapy with SDF-1 andGM-CSF significantly exceeded those seen in animals receiving G-CSFalone (7-fold versus 3.2-fold above saline treated controls, p<0.01).Since mean numbers of large-lumen vessels at the peri-infarct regionwere similar in animals receiving combined therapy with SDF-1 and GM-CSFand in those receiving G-CSF alone (8.0 vs 7.5/high power field), thesedata suggest that SDF-1 enhanced cardiomyocyte cycling/regeneration byan alternative mechanism in addition to induction of neovascularization.

Intramyocardial injection of SDF-1 improves cardiac function and issynergistic with bone marrow mobilization. Intramyocardial injection ofSDF-1 resulted in a similar degree of functional myocardial recovery aswas seen with systemic G-CSF administration (10% mean improvement inejection fraction between days 2 and 14 post-LAD ligation compared with10% mean improvement for G-CSF treated animals and 17% mean loss forsaline-treated controls, p<0.01 for both treatment arms). Moststrikingly, combining SDF-1 injection with bone marrow mobilization byGM-CSF resulted in significant augmentation in functional recovery (21%mean improvement in ejection fraction, p<0.01), FIG. 20( b). These dataindicate that intramyocardial administration of SDF-1 causes improvementin cardiac function after acute ischemia through two separatemechanisms, a direct mechanism which involves induction of cardiomyocytecycling and regeneration and an indirect mechanism operating throughenhanced chemotaxis of mobilized bone marrow-derived endothelialprogenitors and cardiac neovascularization.

We further investigated the role of SDF as an agent for inducing cardiacregeneration. We found that CXCR4 expression occurs following acuteinduced myocardial ischemia in focal and peri-infarct areas (see FIG.26). In separate experiments, we found that intramyocardial injection ofSDF-1 induced early phosphorylation of AKT (see FIG. 27). Furthermore,in cultured rat neonatal cardiomyocytes (which also express CXCR4, seeFIG. 28), SDF administration induced phosphorylation of both AKT and ERKin a time-dependent manner. FIG. 29 shows rapid phosphorylation of AKTin cultured myocytes in the presence of SDF-1. Consistent with theseresults, further investigation demonstrated that intracardiacadministration of SDF-1 augments G-CSF-induced neovascularization andregeneration of acutely ischemic myocardium (see FIG. 30). In addition,intracardiac administration of SDF-1 was also found to augmentG-CSF-induced functional myocardial recovery following myocardialischemia in rats (see FIG. 31), as well as protect rat neonatal cardiacmyocytes against H₂O₂ apopotsis in a dose dependent manner (FIG. 32).

DISCUSSION

Exogenous SDF may induce tissue repair via trophic effects on local orbone-marrow derived progenitors. In addition, increasing SDF-1 in theheart after ischemic insult may result in trophic effects oncardiomyocyte progenitors and induce cardiac repair and regeneration.

Methods and Materials Purification and Characterization ofCytokine-Mobilized Human CD34+ Cells:

Single-donor leukopheresis products were obtained from humans treatedwith recombinant G-CSF 10 mg/kg (Amgen, Calif.) sc daily for four days.Donors were healthy individuals undergoing standard institutionalprocedures of bone marrow mobilization, harvesting and isolation forallogeneic stem cell transplants. Mononuclear cells were separated byFicoll-Hypaque, and highly-purified CD34+ cells (>98% positive) wereobtained using magnetic beads coated with anti-CD34 monoclonal antibody(mAb) (Miltenyi Biotech, CA). Purified CD34 cells were stained withfluorescein-conjugated mAbs against CD34 and CD117 (Becton Dickinson,CA), AC133 (Miltenyi Biotech, CA), CD54 (Immunotech, CA), CD62E(BioSource, MA), VEGFR-2, Tie-2, vWF, eNOS, CXCR1, CXCR2, and CXCR4 (allSanta Cruz Biotech, CA), and analyzed by four-parameter fluorescenceusing FACScan (Becton Dickinson, CA). Cells positively selected for CD34expression were also stained with phycoerythrin (PE)-conjugatedanti-CD117 mAb (Becton Dickinson, CA), and sorted for bright and dimfluorescence using a Facstar Plus (Becton Dickinson) and a PE filter.Intracellular staining for GATA-2 was performed by permeabilizing onemillion cells from each of the brightly and dimly fluorescing cellpopulations using a Pharmingen Cytofix/Cytoperm™ kit, incubating for 30minutes on ice with 10 μl of fluorochrome-conjugated mAbs against bothCD117 and CD34 surface antigens (Becton Dickinson, CA). Afterresuspension in 250 μl of Cytofix/Cytoperm™ solution for 20 minutes at 4degrees C., cells were incubated with a fluorochrome-labeled mAb againstGATA-2 (Santa Cruz Biotech, CA) or IgG control for 30 minutes at 4degrees C., and analyzed by three-parameter flow cytometry.

Chemotaxis of Human Bone-Marrow Derived Endothelial Progenitors:

Highly-purified CD34+CD117^(bright) cells (>98% purity) were plated in48-well chemotaxis chambers fitted with membranes (8 mm pores) (NeuroProbe, MD). After incubation for 2 hours at 37° C., chambers wereinverted and cells were cultured for 3 hours in medium containing IL-8,SDF-1 alpha/beta, and SCF at 0.2, 1.0 and 5.0 μg/ml. The membranes werefixed with methanol and stained with Leukostat™ (Fischer Scientific,Ill). Chemotaxis was calculated by counting migrating cells in 10high-power fields.

Animals, Surgical Procedures, Injection of Human Cells, and Quantitationof Cellular Migration into Tissues:

Rowett (rnu/rnu) athymic nude rats (Harlan Sprague Dawley, Indianapolis,Ind.) were used in studies approved by the “Columbia UniversityInstitute for Animal Care and Use Committee”. After anesthesia, a leftthoracotomy was performed, the pericardium was opened, and the leftanterior descending (LAD) coronary artery was ligated. Sham-operatedrats had a similar surgical procedure without having a suture placedaround the coronary artery. For studies on cellular migration, 2.0×10⁶CD34+ cells obtained from a single donor after G-CSF mobilization wereinjected into the tail vein 48 hours after LAD ligation either alone ortogether with 50 μg/ml monoclonal antibody (mAb) with known functionalinhibitory activity against either human CXCR1, human CXCR2, humanCXCR4, rat SDF-1 (all R & D Systems, MN), human CD34 (Pharmingen, CA),or rat IL-8 (ImmunoLaboratories, Japan). Controls received eitherisotype control antibodies at the same concentration or saline after LADligation. Prior to injection, 2.0×10⁶ human cells were incubated with2.5 ug/mL of the fluorescent carbocyanine DiI dye (Molecular Probes) for5 minutes at 37° C. and 15 minutes at 4° C. After washing in PBS,DiI-labeled human cells were resuspended in saline and injectedintravenously. 2.0×10⁶ CD34+ human cells were also injected into thetail vein of sham-operated or LAD-ligated rats receiving threeintramyocardial injections of 1.0 μg/ml recombinant human IL-8, SDF-1,SCF or saline. Each group consisted of 6-10 rats. Quantitation ofmyocardial infiltration after injection of human cells was performed byassessment of DiI fluorescence in hearts from rats sacrificed 2 daysafter injection (expressed as number of DiI-positive cells per highpower field, minimum 5 fields examined per sample). Quantitation of ratbone marrow infiltration by human cells was performed in 12 rats atbaseline, days 2, 7, and 14 by flow cytometric and RT-PCR analysis ofthe proportion of HLA class I-positive cells relative to the total ratbone marrow population. For studies on neoangiogenesis and effects onmyocardial viability and function, 2.0×10⁶ DiI-labelled human CD34+cells obtained from a single donor after G-CSF mobilization werereconstituted with 10³, 10⁵, or 2.0×10⁵ immunopurifiedCD34+CD117^(bright) cells, and injected into the rat tail vein 48 hoursafter LAD ligation, in the presence or absence of a mAb with knowninhibitory activity against CXCR4. Each group consisted of 6-10 rats.Histologic and functional studies were performed at 2 and 15 weeks.

Measurement of Rat CXC Chemokine mRNA and Protein Expression:

Poly(A)+ mRNA was extracted by standard methods from the hearts of 3normal and 12 LAD-ligated rats. RT-PCR was used to quantify myocardialexpression of rat IL-8 and Gro-alpha mRNA at baseline and at 6, 12, 24and 48 hours after LAD ligation after normalizing for total rat RNA asmeasured by GAPDH expression. After priming with oligo (dT) 15-mer andrandom hexamers, and reverse transcribed with Moloney murinelymphotrophic virus reverse transcriptase (Invitrogen, Carlsbad, Calif.,USA), cDNA was amplified in the polymerase chain reaction (PCR) usingTaq polymerase (Invitrogen, Carlsbad, Calif., USA), radiolabeleddideoxy-nucleotide ([a32P]-ddATP: 3,000 Ci/mmol, Amersham, ArlingtonHeights, Ill.), and primers for rat Cinc (rat homologue of humanIL-8/Gro-alpha and GAPDH (Fisher Genosys, CA). Primer pairs(sense/antisense) for rat Cinc and GAPDH were, gaagatagattgcaccgatg (SEQID NO:4)/catagcctctcacatttc SEQ ID NO:5),gcgcccgtccgccaatgagctgcgc SEQID NO:6)/cttggggacacccttcagcatcttttgg SEQ ID NO:7), andctctacccacggcaagttcaa SEQ ID NO:8)/gggatgaccttgcccacagc SEQ ID NO:9),respectively. The labelled samples were loaded into 2% agarose gels,separated by electrophoresis, and exposed for radiography for 6 h at−70° C. Serum levels of rat IL-8/Gro-alpha were measured at baseline andat 6, 12, 24 and 48 hours after LAD ligation in four rats by acommercial ELISA using polyclonal antibodies against the rat IL-8/Grohomologue Cinc (ImmunoLaboratories, Japan). The amount of protein ineach serum sample was calculated according to a standard curve ofoptical density (OD) values constructed for known levels of ratIL-8/Gro-alpha protein. Anti-Cinc antibodies were also used according tothe manufacturer's instructions at 1:200 dilution in immunohistochemicalstudies to identify the cellular source of Cinc production in ratmyocardium after LAD ligation. Positively-staining cells were visualizedas dark gray through the Avidin/Biotin system described below.

Histology and Measurement of Infarct Size:

Following excision at 2 and 15 weeks, left ventricles from eachexperimental animal were sliced at 10-15 transverse sections from apexto base. Representative sections were fixed in formalin and stained forroutine histology (H&E) to determine cellularity of the myocardium,expressed as cell number per high power field (HPF) (600×). A Massontrichrome stain was performed, which labels collagen blue and myocardiumred, to evaluate collagen content on a semiquantitative scale (0-3+),with 1+ light blue, 2+ light blue and patches of dark blue, and 3+ darkblue staining. This enabled measurement of the size of the myocardialscar using a digital image analyzer. The lengths of the infarctedsurfaces, involving both epicardial and endocardial regions, weremeasured with a planimeter digital image analyzer and expressed as apercentage of the total ventricular circumference. Final infarct sizewas calculated as the average of all slices from each heart. All studieswere performed by a blinded pathologist. Infarct size was expressed aspercent of total left ventricular area. Final infarct size wascalculated as the average of all slices from each heart.

Quantitation of Capillary Density:

In order to quantitate capillary density and species origin of thecapillaries, additional sections were stained freshly with mAbs directedagainst rat or human CD31 (Serotec, UK, and Research Diagnostics, NJ,respectively), factor VIII (Dako, CA), and rat or human MHC class I(Accurate Chemicals, CT). Arterioles were differentiated from largecapillaries by the presence of a smooth muscle layer, identified bystaining sections with a monoclonal antibody against muscle-specificdesmin (Dako, Ca). Staining was performed by immunoperoxidase techniqueusing an Avidin/Biotin Blocking Kit, a rat-absorbed biotinylatedanti-mouse IgG, and a peroxidase-conjugate (all Vector LaboratoriesBurlingame, Calif.). Capillary density was determined at 2 weeks postinfarction from sections labeled with anti-CD31 mAb, and confirmed withanti-factor VIII mAb, and compared to the capillary density of theunimpaired myocardium. Values are expressed as the number ofCD31-positive capillaries per HPF (400×).

Quantitation of Cardiomyocyte Proliferation:

Cardiomyocyte DNA synthesis and cell cycling was determined by dualstaining of rat myocardial tissue sections obtained from LAD-ligatedrats at two weeks after injection of either saline or CD34+ human cells,and from healthy rats as negative controls, for cardiomyocyte-specifictroponin I and human- or rat-specific Ki-67. Briefly, paraffin embeddedsections were microwaved in a 0.1M EDTA buffer, and stained with eithera primary monoclonal antibody against rat Ki-67 at 1:3000 dilution (giftof Giorgio Catoretti, Columbia University) or human Ki-67 at 1:300dilution (Dako, CA) and incubated overnight at 4 degrees C. Followingwashes, sections were incubated with a species-specific secondaryantibody conjugated with alkaline phosphatase at 1:200 dilution (VectorLaboratories Burlingame, Calif.) for 30 minutes and positively-stainingnuclei were visualized as blue with a BCIP/NBT substrate kit (Dako, CA).Sections were then incubated overnight at 4 degrees C. with a monoclonalantibody against cardiomyocyte-specific troponin I (Accurate Chemicals,CT) and positively-staining cells were visualized as dark gray throughthe Avidin/Biotin system described above. Cardiomyocytes progressingthrough cell cycle in the infarct zone, peri-infarct region, and areadistal to the infarct were calculated as the proportion of troponinI-positive cells per high power field co-expressing Ki-67.

Measurement of Myocyte Apoptosis by DNA End-Labeling of Paraffin TissueSections:

For in situ detection of apoptosis at the single cell level we used theTUNEL method of DNA end-labeling mediated by dexynucleotidyl transferase(TdT) (Boehringer Mannheim, Mannheim, Germany). Rat myocardial tissuesections were obtained from LAD-ligated rats at two weeks afterinjection of either saline or CD34+ human cells, and from healthy ratsas negative controls. Briefly, tissues were deparaffinized with xyleneand rehydrated with graded dilutions of ethanol and two washes inphosphate-buffered saline (PBS). The tissue sections were then digestedwith Proteinase K (10 μg/ml in Tris/HCL) for 30 minutes at 37° C. Theslides were then washed 3 times in PBS and incubated with 50 μl of theTUNEL reaction mixture (TdT and fluorescein-labeled dUTP) and incubatedin a humid atmosphere for 60 minutes at 37° C. For negative controls TdTwas eliminated from the reaction mixture. Following 3 washes in PBS, thesections were then incubated for 30 minutes with an antibody specificfor fluorescein-conjugated alkaline phosphatase (AP) (BoehringerMannheim, Mannheim, Germany). The TUNEL stain was visualized with asubstrate system in which nuclei with DNA fragmentation stained blue,(BCIP/NBT substrate system, Dako, Carpinteria, Calif.). The reaction wasterminated following three minutes of exposure with PBS. To determinethe proportion of blue-staining apoptotic nuclei within myocytes, tissuewas counterstained with a monoclonal antibody specific for desmin.Endogenous peroxidase was blocked by using a 3% hydrogen perioxidasesolution in PBS for 15 minutes, followed by washing with 20% goat serumsolution. An anti-troponin I antibody (Accurate Chemicals, CT) wasincubated overnight (1:200) at 40 degrees C. Following 3 washes sectionswere then treated with an anti-rabbit IgG, followed by a biotinconjugated secondary antibody for 30 minutes (Sigma, Saint Louis, Mo.).An avidin-biotin complex (Vector Laboratories, Burlingame, Calif.) wasthen added for an additional 30 minutes and the myocytes were visualizeddark gray following 5 minutes exposure in DAB solution mixture (Sigma,Saint Louis, Mo.). Tissue sections were examined microscopically at 200×magnification. Within each 200× field 4 regions were examined,containing at least 250 cells per region and cumulatively approximating1 mm² of tissue, at both the peri-infarct site and distally to thissite. Stained cells at the edges of the tissue were not counted. Resultswere expressed as the mean number of apoptotic myocytes per mm² at eachsite examined.

Analysis of Myocardial Function:

Echocardiographic studies were performed using a high frequency linerarray transducer (SONOS 5500, Hewlett Packard, Andover, Mass.). 2Dimages were obtained at mid-papillary and apical levels. End-diastolic(EDV) and end-systolic (ESV) left ventricular volumes were obtained bybi-plane area-length method, and % left ventricular ejection fractionwas calculated as [(EDV−ESV)/EDV]×100.

CDNA Subtractive Hybridization:

Briefly, messenger RNA was isolated from each heart, and 1 μg was usedfor first-strand cDNA synthesis with random primers. The subtractivehybridization was performed with the PCR-select cDNA subtraction kit(CLONTECH), following the manufacturer's recommendations. Aftersecond-strand synthesis, the two cDNA libraries were digested with RsaI.Digestion products of the “tester” library were ligated to a specificadapter (T7 promoter), then hybridized with a 30-fold excess of the“driver” library for subtraction. After hybridization, the remainingproducts were further amplified by PCR. In the forward subtraction,which determines the genes that are overexpressed in the ischemicsample, the ischemic tissue is the “tester” and the normal tissue is the“driver.” In the reverse subtraction, the “tester” and the “driver” areswitched to determine the genes that are down-regulated in the ischemicsample.

Rat Neonatal Cardiac Myocyte Culture:

Neonatal cardiac myocytes were isolated from ventricles of 1 to 3-dayold Sprague Dawley rats. Myocytes were isolated by step-wise enzymaticdigestion using a modification of a previously described method(Sadoshima et al., 1992, J. Biol. Chem. 267:10551-10560). PERCOLLgradient was used to enrich for cardiac myocytes during isolation.Myocytes were plated onto gelatin-coated dishes or chamber slides(LABTEK PERMANOX, Nunc) in DMEM/F12 media containing 5% horse serum.Sixteen hours following plating cells were washed and serum starved ifrequired.

Protein Isolation: Rat Isolated Ventricle.

Isolated ventricles were frozen in liquid nitrogen and stored at −80° C.Frozen ventricle were Polytron homogenised on ice (3 times, 10-s bursts)in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mMEDTA, 1 nM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM Na₃VO₄ and 1mM NaF. NP-40 (final concentration 1%) was added for 30 min, thensamples were Dounce homogenized on ice (60 strokes). Followingcentrifugation (6000 rpm, 15 min) the protein concentration of thesupernatant was determined (BIORAD Protein Assay).

Neonatal Rat Cardiac Myocytes.

Myocytes were washed twice with ice-cold PBS before collection in RIPAbuffer containing the above described components. Following 15-minincubation on an orbital rocker at 4° C., samples were passed 4 timesthrough a 21G needle. Supernatant was collected after centrifugation(14,000 rpm, 15 min) and protein concentration determined (BIORADProtein Assay).

Western Blot Method: Rat Isolated Ventricle.

Protein extracts (100 μg) prepared in SDS sample buffer were resolved bySDS/PAGE. PVDF membranes were blocked in PBST containing 5% BSA for 1 h,before overnight incubation with anti-phospho AKT or anti-AKT antibodiesdiluted 1:1000 in PBST containing 5% BSA (CELL SIGNALING). Densitometricquantification was performed using IMAGEQUANT software (MolecularDynamics).

Neonatal Rat Cardiac Myocytes.

Serum starved cells were stimulated with SDF-1α at the concentrationsand times described. Protein extracts (50 μg) prepared in SDS samplebuffer were resolved by SDS/PAGE. PVDF membranes were blocked in PBSTcontaining 5% BSA and incubated overnight with anti-phospho AKT oranti-AKT antibodies diluted 1:1000 in PEST containing 5% BSA (CellSignalling). Nitrocellulose membranes were blocked in PBST containing 5%skim milk for 1 h prior to overnight incubation with anti-phospho ERK oranti-ERK antibodies diluted 1:1000 in PBST containing 5% skim milk (CELLSIGNALING). Densitometric quantification was performed using IMAGEQUANTsoftware (Molecular Dynamics).

CXCR4 Immunostaining: Rat Isolated Ventricle.

Cross-sections of ventricle were formalin (10%) fixed and embedded inparaffin. Sections (4 μm) were de-waxed and endogenous peroxide activitywas blocked using 3% H₂O₂ (20 min). After 1 h blocking using swine serum(1:5 dilution, DAKO), sections were incubated overnight with fusin(CXCR4; H-118, Santa Cruz Biotechnology) at a 1:400 dilution.Immunostaining was developed using a biotinylated secondary antibody(DAKO) linked to avidin/HRP (VectaStain Elite ABC Kit, VectorLaboratories) followed by DAB chromagen (DAKO). Slides werecounterstained with haematoxylin.

Neonatal Rat Cardiac Myocytes.

Cells cultured on chamber slides (LabTek Permanox, Nunc) were rinsedwith PBS and fixed with ice-cold methanol for 5 min. Endogenous peroxideactivity was inhibited using 3% H₂O₂ diluted in methanol for 5 min.Slides were blocked for 1 h in swine serum (1:5 dilution, DAKO), andthen incubated overnight with fusin (CXCR4; H-118, Santa CruzBiotechnology) at a 1:200 dilution. Immunostaining was developed using abiotinylated secondary antibody (DAKO) linked to avidin/HRP (VECTASTAINELITE ABC Kit, Vector Laboratories) followed by DAB chromagen (DAKO).

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Experimental Results II

Intravenous Administration of Human Bone Marrow-Derived EndothelialProgenitor Cells Induces Neovascularization and Prevents CardiomyocyteApoptosis within Five Days Post-Infarction.

We sought to determine how soon after intravenous injection of bonemarrow-derived endothelial progenitor cells does neovascularizationdevelop in hearts of rats having undergone permanent LAD ligation 48hours earlier. When animals were sacrificed at two days afterintravenous injection of DiI-labelled human CD34+ cells obtained byG-CSF mobilization (>98% CD34 purity, containing 6-12% CD117brightangioblasts), numerous DiI positive interstitial cells were seen in theperi-infarct region, but no defined vascular structures expressing DiIcould be identified, data not shown. In contrast, animals sacrificed atfive days post-infusion of human CD34+ cells demonstrated numerousDiI-positive vascular structures at the peri-infarct region and 3.5-foldhigher numbers of capillaries in comparison to rats receiving saline,FIG. 21( a) (p<0.01). The increase in microvascularity at 5 days wasaccompanied by 3.3-fold lower numbers of apoptotic cardiomyocytes at theperi-infarct region, defined by dual positivity for troponin I andTUNEL, in comparison to controls receiving saline, FIG. 21( b) (p<0.01).Together, these data indicate that the vasculogenic process ofdifferentiation and organization of bone marrow-derived angioblasts to amature, functional network of capillaries in the ischemic myocardiumtakes from two to five days.

Intravenous Administration of Human Bone Marrow-Derived EndothelialProgenitor Cells Induces Cell Cycling of Cardiomyocyte Progenitors andCardiomyocyte Differentiation.

We examined cardiac tissue from experimental and control animalssacrificed at five days by immunohistochemistry and confocal microscopyfor evidence of cycling cardiomyocytes, as has been suggested to occurrarely in the adult heart after acute ischemia. While no maturetroponin-positive cycling cardiomyocytes were detected in any control orexperimental animals at day 5 post-infarction, animals receiving humanbone marrow-derived CD34+ cells demonstrated numerous clusters of small,cycling cells at the peri-infarct region that were of rat origin, asdefined by a monoclonal antibody specific for rat Ki67, FIG. 22( a).These cells were negative for cardiomyocyte-specific troponin I, amarker of cardiomyocyte differentiation, but stained positively foralpha-sarcomeric actin, indicating they were immature cells ofcardiomyocyte lineage. Similar clusters of cycling cardiomyocyteprogenitors were not seen in control animals injected with saline.

Tissues obtained from animals sacrificed at two weeks after human CD34+administration no longer demonstrated clusters of small, cyclingcardiomyocyte progenitors, but instead a high frequency of large, maturerat cardiomyocytes at the peri-infarct region with detectable DNAactivity, as determined by dual staining with mAbs reactive againstcardiomyocyte-specific troponin I and rat Ki67, FIGS. 22 (b) and (c).The number of cycling, mature cardiomyocytes at the peri-infarct regionwas 4-fold higher in animals receiving human endothelial progenitorcells than in LAD-ligated controls receiving saline (p<0.01) in whomthere was a high frequency of cells with fibroblast morphology andreactivity with rat Ki67, but not troponin I. We speculate that thecycling, mature cardiomyocytes seen at day 14 post-infarction in animalswho received human endothelial progenitor cells are the differentiatedprogeny of the small, cycling immature cardiomyocyte progenitors seen inthe same anatomical location at day 5 and accompanying the onset ofneovascularization. Whether the cycling cardiomyocyte progenitorsrepresent in situ cardiac stem cells normally residing in the heart in aquiescent state, or whether these cells have migrated to the heart fromelsewhere in the body, such as the bone marrow, remains to bedetermined.

Myocardial Expression of HBP23, a Rat Peroxiredoxin Protecting CellsAgainst Damage by Oxygen Radicals, is Decreased by Ischemia andIncreased by Neovascularization.

We next sought to identify a molecular mechanism to explain therelationship between neovascularization at the peri-infarct region andproliferation/regeneration of adjacent cardiomyocyte progenitors. Wefirst performed cDNA subtractive hybridization in order to identifypatterns of changes in gene expression between normal rat hearts and rathearts 48 hours after LAD ligation. Since deficits in antioxidants andincreased oxidative stress accompanying myocardial infarction have beendirectly implicated in the pathogenesis of post-infarct heart failure(13-15), we chose to examine changes in expression of particularantioxidant genes. A family of antioxidant enzymes known asperoxiredoxins, which demonstrate peroxidase activity (16) and areinduced by oxygen (17), play a critical role in regulating cell survivalduring periods of oxidative stress, such as ischemia. They serve tomaintain the thiol-disulfide status of the cytosol by undergoingreversible oxidation-reduction reactions involving electron transfer viadisulfide bridges formed with thioredoxin (TRX), which constitutes oneof the principal cellular reducing systems in mammals (18). In addition,peroxiredoxins can inhibit c-Abl tyrosine kinase activity induced byoxidative stress (19,20) and can rescue cells from both a pro-apoptoticstate and cell cycle arrest induced by the activated c-Abl gene product(21). By cDNA subtractive hybridization, we found that expression of therat peroxiredoxin HBP23 mRNA was reduced in rat hearts 48 hours afterLAD ligation compared with normal rat hearts. By RT-PCR, HBP23 mRNAlevels in rat hearts decreased at two weeks post-LAD ligation by a meanof 34% compared with normal rat hearts, FIG. 23 (p<0.01). In contrast,HBP23 mRNA levels in LAD-ligated rat hearts two weeks after receivinghuman angioblasts returned to levels only 14% lower than in non-ischemiccontrols. Since mRNA expression of peroxiredoxins is induced by oxygen,these data suggested that HBP23 mRNA expression was inhibited by theacute ischemic event and induced following angioblast-dependentneovascularization.

Generation Of A DNA Enzyme To Cleave HBP23 mRNA.

To investigate whether induced expression of HBP23 is involved in themechanism by which neovascularization affects myocardial cellularapoptosis, regeneration/proliferation, and function, we generated acatalytic DNA enzyme targeting specific sequences within the rat HBP23gene (22,23). We chose to specifically target pyrimidine-purinejunctions at or near the translational start site AUG of messenger RNAfor rat HBP23, a region that is conserved between species and has lowrelative free energy (24). In this region, the rat HBP23 sequencediffers by only one base from the human homologue,proliferation-associated gene (PAG). To produce the control DNA enzyme,the nucleotide sequence in the two flanking arms of the HBP23 DNA enzymewas scrambled without altering the catalytic domain. The 3′ terminus ofeach molecule was capped with an inverted 3′-3′-linked thymidine forresistance to 3′-to-5′ exonuclease digestion.

The DNA enzyme against HBP23 cleaved the 23-base oligonucleotidesynthesized from the sequence of rat HBP23 mRNA, in a dose- andtime-dependent manner, FIG. 24 (a). In contrast, the DNA enzyme did notcleave a 23-base oligonucleotide form the human homologue PAG whichdiffers from the rat HBP23 oligonucleotide by only one base,demonstrating its exquisite target specificity. A DNA enzyme withspecificity for the same translational start site in the human PAG geneefficiently cleaved the PAG oligonucleotide, but not the one derivedfrom HBP23 (data not shown). The scrambled control DNA enzyme did notcleave either oligonucleotide. To determine the effect of the DNA enzymeon endogenous HBP23 production, cardiomyocyte monolayers obtained fromfetal rat hearts were grown to confluence and transfected withspecies-specific DNA enzymes or scrambled control. Densitometricanalysis of RT-PCR products following reverse transcription of cellularmRNA showed that the HBP23 DNA enzyme inhibited steady-state mRNA levelsin cultured rat cells by over 80%, FIG. 24 (b), relative to thescrambled DNA.

In Vivo Administration of a DNA Enzyme to Prevent Induction of HBP23mRNA in Rat Myocardium: Neovascularization is not Affected, but itsEffects on Cardiomyocyte Apoptosis, Regeneration, and Function areAbrogated.

To investigate the in vivo relevance of induced expression of HBP23 inexperimental myocardial infarction, 48 hours after LAD ligation ratswere injected with human bone marrow-derived endothelial progenitorcells intravenously together with intramyocardial injections of eitherHBP23 DNA enzyme or scrambled control. As shown in FIG. 25 (a), HBP23DNA enzyme had no effect on induction of neovascularization by humanbone marrow angioblasts. When sacrificed at day 5 post-infusion, ratswho received human endothelial progenitors, irrespective of whetherHBP23 DNA enzyme or scrambled control was co-injected, demonstratedincreased myocardial capillary density in comparison to saline controls.In contrast, injection of the HBP23 DNA enzyme, but not the scrambledcontrol, abrogated the anti-apoptotic effects of neovascularization,FIG. 25 (b), and the improvement in cardiac function, FIG. 25 (c). Amonganimals receiving human endothelial progenitor cells and demonstratingmyocardial neovascularization, treatment with HBP23 DNA enzyme resultedin 1.7-fold higher levels of cardiomyocyte apoptosis (p<0.01) and 38%mean deterioration in cardiac function as assessed by echocardiography(p<0.01). In addition, the HBP23 DNA enzyme exerted striking effects oncardiomyocyte progenitor proliferation/regeneration. Whereasperi-infarct clusters of small rat cells expressing Ki67 andalpha-sarcomeric actin were easily detected in animals receivingangioblasts together with the scrambled control DNA, none wereidentified in animals receiving the HBP23 DNA enzyme. These resultsclearly demonstrate that angioblast-dependent neovascularizationprotects cardiomyocytes against apoptosis and inducesproliferation/regeneration of resident cardiomyocyte lineage progenitorsvia pathways regulated by peroxiredoxin gene products.

DISCUSSION

In this study we showed that neovascularization of ischemic myocardiumby human bone marrow derived angioblasts results in both protection ofmature cardiomyocytes at the peri-infarct region against apoptosis andstimulation of cardiomyocyte progenitors at the same site to enter cellcycle, proliferate and regenerate. Moreover, we showed that themechanism by which angioblast-dependent neovascularization regulatescardiomyocyte survival and self-renewal appears to involve at least onefamily of genes involved in anti-apoptotic and pro-proliferativepathways accompanying oxidative stress, the peroxiredoxins. While oxygentension positively regulates peroxiredoxin gene expression (17), whetherthis alone explains the positive effect of angioblast-mediatedneovascularization on HBP mRNA expression is not clear. Sinceperoxiredoxin mRNA expression is induced by protein kinase C delta (25)it is possible that bone marrow angioblasts may be a source ofextracellular signals regulating protein kinase C delta activation andconsequently cell survival, such as FGF-1 (26).

Deficits in antioxidants and increased oxidative stress accompanyingmyocardial infarction have been directly implicated in the pathogenesisof post-infarct heart failure (13-15). Our study suggests that reducedlevels of peroxiredoxins post-infarction may be of direct causality inthe subsequent remodelling and failure of the left ventricle. Theantioxidant effects of peroxiredoxins are coupled with the physiologicalelectron donor activity of the TRX system (27-29). In addition todirectly interacting with TRX, the peroxiredoxin gene productsspecifically bind the SH3 domain of c-Abl, a non-receptor tyrosinekinase, inhibiting its activation (21). Activation of c-Abl through theSH3 domain by stimuli such as agents that damage DNA induces eitherarrest of the cell cycle in phase G1 or cellular apoptosis (30). Cellcycle arrest is dependent on the kinase activity of c-Abl whichdownregulates the activity of the cyclin-dependent kinase Cdk2 andinduces the expression of p21 (31). By associating with c-Abl in vivo,peroxiredoxins can inhibit tyrosine phosphorylation induced by c-Abloverexpression and rescue cells from both the cytostatic andpro-apoptotic effects of the activated c-Abl gene product (32). Our invivo demonstration that co-administration of a DNA enzyme directedagainst the rat peroxiredoxin HBP23 abrogated the anti-apoptotic andpro-proliferative effects of human angioblast-dependentneovascularization in the infarcted rat heart argues strongly that thisfamily of genes is directly implicated in the mechanism of action bywhich neovascularization results in improvement in cardiac function andprevention of heart failure.

Throughout life, a mixture of young and old cells is present in thenormal myocardium. Although most myocytes seem to be terminallydifferentiated, there is a fraction of younger myocytes that retains thecapacity to replicate (33). In the present study, humanangioblast-dependent neovascularization resulted, Within five days, inproliferation of small, endogenous rat cardiomyocyte precursors at theperi-infarct region. The dividing myocyte precursors could be identifiedby immunohistochemical criteria on the basis of concomitant cell surfaceexpression of alpha-sarcomeric actin, but not troponin I, andproliferating nuclear structures, defined by an antibody specific forrat Ki67. Since this process was followed within fourteen days byincreasing numbers in the same location of mature, cyclingcardiomyocytes, defined on the basis of morphology, cell surfaceexpression of troponin I, and nuclear expression of Ki67, we concludethat cycling cardiomyocyte precursors differentiated in situ to becomenew, mature, functional cardiomyocytes. Whether these precursors arederived from a resident pool of cardiomyocyte stem cells or from arenewable source of circulating bone marrow-derived stem cells that hometo the damaged myocardium remains to be determined. Moreover, while thesignals required for in situ expansion of cardiomyocyte precursorsappear to involve, at least in part, pathways regulated by members ofthe peroxiredoxin gene family, the signals required for cardiomyocytedifferentiation are, at present, unknown. Gaining an understanding ofthese issues may open the possibility of manipulating the biology ofendogenous cardiomyocytes in order to augment the healing process aftermyocardial ischemia.

Methods and Materials Purification and Characterization ofCytokine-Mobilized Human CD34+ Cells.

Single-donor leukopheresis products were obtained from humans treatedwith recombinant G-CSF 10 mg/kg (Amgen, Calif.) sc daily for four days.Donors were healthy individuals undergoing standard institutionalprocedures of bone marrow mobilization, harvesting and isolation forallogeneic stem cell transplants. Mononuclear cells were separated byFicoll-Hypaque, and highly-purified CD34+ cells (>98% positive) wereobtained using magnetic beads coated with anti-CD34 monoclonal antibody(mAb) (Miltenyi Biotech, CA). Purified CD34 cells were stained withfluorescein-conjugated mAbs against CD34 and CD117 (Becton Dickinson,CA), AC133 (Miltenyi Biotech, CA), CD54 (Immunotech, CA), CD62E(BioSource, MA), VEGFR-2, Tie-2, vWF, eNOS, CXCR1, CXCR2, and CXCR4 (allSanta Cruz Biotech, CA), and analysed by four-parameter fluorescenceusing FACScan (Becton Dickinson, CA). Cells positively selected for CD34expression were also stained with phycoerythrin (PE)-conjugatedanti-CD117 mAb (Becton Dickinson, CA), and sorted for bright and dimfluorescence using a Facstar Plus (Becton Dickinson) and a PE filter.Intracellular staining for GATA-2 was performed by permeabilizing onemillion cells from each of the brightly and dimly fluorescing cellpopulations using a Pharmingen Cytofix/Cytoperm kit, incubating for 30minutes on ice with 10 ul of fluorochrome-conjugated mAbs against bothCD117 and CD34 surface antigens (Becton Dickinson, CA). Afterresuspension in 250 ul of Cytofix/Cytoperm solution for 20 minutes at 4degrees C., cells were incubated with a fluorochrome-labeled mAb againstGATA-2 (Santa Cruz Biotech, CA) or IgG control for 30 minutes at 4degrees C., and analyzed by three-parameter flow cytometry.

Animals, Surgical Procedures, and Injection of Human Cells.

Rowett (rnu/rnu) athymic nude rats (Harlan Sprague Dawley, Indianapolis,Ind.) were used in studies approved by the “Columbia UniversityInstitute for Animal Care and Use Committee”. After anesthesia, a leftthoracotomy was performed, the pericardium was opened, and the leftanterior descending (LAD) coronary artery was ligated. Sham-operatedrats had a similar surgical procedure without having a suture placedaround the coronary artery.

Histology and Measurement of Infarct Size.

Following excision at 2 and 15 weeks, left ventricles from eachexperimental animal were sliced at 10-15 transverse sections from apexto base. Representative sections were fixed in formalin and stained forroutine histology (H&E) to determine cellularity of the myocardium,expressed as cell number per high power field (HPF) (600×). A Massontrichrome stain was performed, which labels collagen blue and myocardiumred, to evaluate collagen content on a semiquantitative scale (0-3+),with 1+ light blue, 2+ light blue and patches of dark blue, and 3+ darkblue staining. This enabled measurement of the size of the myocardialscar using a digital image analyzer. The lengths of the infarctedsurfaces, involving both epicardial and endocardial regions, weremeasured with a planimeter digital image analyzer and expressed as apercentage of the total ventricular circumference. Final infarct sizewas calculated as the average of all slices from each heart. All studieswere performed by a blinded pathologist (MJS). Infarct size wasexpressed as percent of total left ventricular area. Final infarct sizewas calculated as the average of all slices from each heart.

Quantitation of Capillary Density.

In order to quantitate capillary density and species origin of thecapillaries, additional sections were stained freshly with mAbs directedagainst rat or human CD31 (Serotec, UK, and Research Diagnostics, NJ,respectively), factor VIII (Dako, CA), and rat or human MHC class I(Accurate Chemicals, CT). Arterioles were differentiated from largecapillaries by the presence of a smooth muscle layer, identified bystaining sections with a monoclonal antibody against muscle-specificdesmin (Dako, Ca). Staining was performed by immunoperoxidase techniqueusing an Avidin/Biotin Blocking Kit, a rat-absorbed biotinylatedanti-mouse IgG, and a peroxidase-conjugate (all Vector LaboratoriesBurlingame, Calif.). Capillary density was determined at 5 days and 2weeks post infarction from sections labeled with anti-CD31 mAb, andconfirmed with anti-factor VIII mAb, and compared to the capillarydensity of the unimpaired myocardium. Values are expressed as the numberof CD31-positive capillaries per HPF (400×).

Quantitation of Cardiomyocyte Proliferation.

Cardiomyocyte DNA synthesis and cell cycling was determined by dualstaining of rat myocardial tissue sections obtained from LAD-ligatedrats at two weeks after injection of either saline or CD34+ human cells,and from healthy rats as negative controls, for cardiomyocyte-specifictroponin I and human- or rat-specific Ki67. Briefly, paraffin embeddedsections were microwaved in a 0.1M EDTA buffer, and stained with eithera primary monoclonal antibody against rat Ki67 at 1:3000 dilution (giftof Giorgio Catoretti, Columbia University) or human Ki67 at 1:300dilution (Dako, CA) and incubated overnight at 4 degrees C. Followingwashes, sections were incubated with a species-specific secondaryantibody conjugated with alkaline phosphatase at 1:200 dilution (VectorLaboratories Burlingame, Calif.) for 30 minutes and positively-stainingnuclei were visualized as blue with a BCIP/NBT substrate kit (Dako, CA).Sections were then incubated overnight at 4 degrees C. with a monoclonalantibody against cardiomyocyte-specific troponin I (Accurate Chemicals,CT) and positively-staining cells were visualized as brown through theAvidin/Biotin system described above. Cardiomyocytes progressing throughcell cycle in the infarct zone, peri-infarct region, and area distal tothe infarct were calculated as the proportion of troponin I-positivecells per high power field co-expressing Ki67. For confocal microscopy,fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG wasused as secondary antibody to detect Ki67 in nuclei. A Cy5-conjugatedmouse mAb against alpha-sarcomeric actin (clone 5C5; Sigma) was used todetect cardiomyocytes, and propidium iodide was used to identify allnuclei.

Measurement of Myocyte Apoptosis by DNA End-Labeling of Paraffin TissueSections.

For in situ detection of apoptosis at the single cell level we used theTUNEL method of DNA end-labeling mediated by dexynucleotidyl transferase(TdT) (Boehringer Mannheim, Mannheim, Germany). Rat myocardial tissuesections were obtained from LAD-ligated rats at two weeks afterinjection of either saline or CD34+ human cells, and from healthy ratsas negative controls. Briefly, tissues were deparaffinized with xyleneand rehydrated with graded dilutions of ethanol and two washes inphosphate-buffered saline (PBS). The tissue sections were then digestedwith Proteinase K (10 ug/ml in Tris/HCL) for 30 minutes at 370 C. Theslides were then washed 3 times in PBS and incubated with 50 ul of theTUNEL reaction mixture (TdT and fluorescein-labeled dUTP) and incubatedin a humid atmosphere for 60 minutes at 370 C. For negative controls TdTwas eliminated from the reaction mixture. Following 3 washes in PBS, thesections were then incubated for 30 minutes with an antibody specificfor fluorescein-conjugated alkaline phosphatase (AP) (BoehringerMannheim, Mannheim, Germany). The TUNEL stain was visualized with asubstrate system in which nuclei with DNA fragmentation stained blue,(BCIP/NBT substrate system, DAKO, Carpinteria, Calif.). The reaction wasterminated following three minutes of exposure with PBS. To determinethe proportion of blue-staining apoptotic nuclei within myocytes, tissuewas counterstained with a monoclonal antibody specific for desmin.Endogenous peroxidase was blocked by using a 3% hydrogen perioxidasesolution in PBS for 15 minutes, followed by washing with 20% goat serumsolution. An anti-troponin I antibody (Accurate Chemicals, CT) wasincubated overnight (1:200) at 40 degrees C. Following 3 washes sectionswere then treated with an anti-rabbit IgG, followed by a biotinconjugated secondary antibody for 30 minutes (Sigma, Saint Louis, Mo.).An avidin-biotin complex (Vector Laboratories, Burlingame, Calif.) wasthen added for an additional 30 minutes and the myocytes were visualizedbrown following 5 minutes exposure in DAB solution mixture (Sigma, SaintLouis, Mo.). Tissue sections were examined microscopically at 20×magnification. Within each 20× field 4 regions were examined, containingat least 250 cells per region and cumulatively approximating 1 mm2 oftissue, at both the peri-infarct site and distally to this site. Stainedcells at the edges of the tissue were not counted. Results wereexpressed as the mean number of apoptotic myocytes per mm2 at each siteexamined.

Analyses of Myocardial Function:

Echocardiographic studies were performed using a high frequency linerarray transducer (SONOS 5500, Hewlett Packard, Andover, Mass.). 2Dimages were obtained at mid-papillary and apical levels. End-diastolic.(EDV) and end-systolic (ESV) left ventricular volumes were obtained bybi-plane area-length method, and % left ventricular ejection fractionwas calculated as [(EDV−ESV)/EDV]×100.

cDNA Subtractive Hybridization:

This technique enabled comparison of the pattern of gene expressionbetween hearts from normal rats and rats who underwent left anteriordescending (LAD) coronary artery ligation 48 hours earlier. Briefly,messenger RNA was isolated from each heart, and 1 μg was used forfirst-strand cDNA synthesis with random primers. The subtractivehybridization was performed with the PCR-select cDNA subtraction kit(CLONTECH), following the manufacturer's recommendations. Aftersecond-strand synthesis, the two cDNA libraries were digested with RsaI.Digestion products of the “tester” library were ligated to a specificadapter (T7 promoter), then hybridized with a 30-fold excess of the“driver” library for subtraction. After hybridization, the remainingproducts were further amplified by PCR. In the forward subtraction,which determines the genes that are overexpressed in the ischemicsample, the ischemic tissue is the “tester” and the normal tissue is the“driver.” In the reverse subtraction, the “tester” and the “driver” areswitched to determine the genes that are down-regulated in the ischemicsample.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis ofHBP23 mRNA Expression:

Total RNA was extracted using RNeasy Kits from Qiagen (Valencia, Calif.)from normal rat hearts or from hearts of rats who underwent LAD ligationtwo weeks earlier and received either saline or human angioblasts. RNAwas reverse transcribed with SMART cDNA Synthesis Kit (Clontech, PaloAlto, Calif.). Amplification reactions were conducted in a 25 ul volumewith an initial step of 94 C for 5 min, followed by 26-32 cycles of 94 Cfor 30 sec and 68 C for 1 min, using TITANIUM Taq PCR Kits (Clontech,Palo Alto, Calif.). Primers for HBP23 were5′-GCTGATGAAGGTATCTCTTTCAGGGGCCTC (SEQ ID NO:10) and5′-GATGGTCTGCCCCTTACCAATAGTGGAAG (SEQ ID NO:11). Rat GAPDH was used asinternal control (forward primer 5′TGAAGGTCGGAGTCAACGGATTTG3′ (SEQ IDNO:12), reverse primer 5′CATGTGGGCCA TGAGG TCCA CCAC3′ (SEQ ID NO:13)).Ethidium bromide stained bands of amplified fragments were quantified bydensitometric.

DNA Enzymes and RNA Substrates:

DNA enzymes with 3′-3′ inverted thymidine were synthesized by IntegratedDNA technologies (Coralville, Iowa) and purified by RNase-free IE-HPLCor RP-HPLC. The short RNA substrates corresponding to target DNA enzymesequences were chemically synthesized followed by RNAse-free PAGEpurification and also made by in vitro transcription from a DNAtemplate. Rat HBP23 cDNA and human PAG cDNA were amplified by RT-PCRfrom total RNA of cultured rat fetal cardiomyocytes and HUVEC,respectively, using the following primer pair:5′TTTACCCTCTTGACTTTACTTTTGTGTGTCCCAC3′ (forward primer) (SEQ ID NO:10)and 5′CCAGCTGGGCACACTTCACCATG3′ (reverse primer) (SEQ ID NO:11). HBP23and PAG cDNA were cloned into pGEM-T vectors (Promega) to obtain plasmidconstructs pGEM-ratHBP23 and pGEM-humanPAG. cDNA sequences were verifiedusing an automatic sequencing machine. 32P-labeled-nucleotide rat HBP23and human PAG RNA transcripts were prepared by in vitro transcription(SP6 polymerase, Promega) in a volume of 20 ml for 1 hour at 32° C.Unincorporated label and short nucleotides (<350base) were separatedfrom radiolabeled species by centrigugation on Chromaspin-200 columns(Clontech, Palo Alto, CA). Synthetic RNA substrates were end-labeledwith 32P using T4 polynucleotide kinase and incubated with 0.05¾5uMHBP23 or scrambled DNA enzyme. Reactions were allowed to proceed at 37°C. and were “quenched” by transfer of aliquots to tubes containing 90%formamide, 20 mM EDTA and loading dye. Samples were separated byelectrophoresis on 15% TBE-urea denaturing polyacrylamide gels anddetected by autoradiography at −80° C. Primary rat fetal cardiomyocyteswere obtained from Clonetic (USA) and grown in medium containing 2% FCS,100 ug/ml streptomycin and 100 IU/ml penicillin at 37° C. in ahumidified atmosphere of 5% CO2. Cells were used in experiments betweenpassage 6 and 8. Subconfluent (70˜80%) rat fetal cardiomyocytes weretransfected using 0.5 ml of serum-free medium containing 0.05%5 uM HBP23or scrambled DNA enzyme and 20 ug/ml cationic lipids (DOTAP). Afterincubation for eight hours cells were lysed using Trizol reagent(LifeSciences, CA) to isolate RNA for RT-PCR of HBP23 expression, asabove.

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1-42. (canceled)
 43. A method of treating a disorder of a subject'sheart involving loss of cardiomyocytes which comprises administering tothe subject a composition comprising an amount of a human stromalderived factor-1, the composition being administered in an amounteffective to cause proliferation of cardiomyocytes within the subject'sheart so as to thereby treat the disorder.
 44. The method of claim 43,wherein the human stromal derived factor-1 is human stromal derivedfactor-1α, human stromal derived factor-1β, or human stromal derivedfactor-1γ.
 45. The method of claim 43, wherein the disorder comprisesmyocardial infarction, congestive heart failure, chronic ischemia, orischemic disease.
 46. The method of claim 43, further comprisingadministering to the subject an amount of one or more of a humangranulocyte-colony stimulating factor, a human granulocytemacrophage-colony stimulating factor, a human interleukin-8, a humanvascular endothelial growth factor, a human fibroblast growth factor, ahuman Gro family chemokine, human endothelial progenitor cells, or apro-angiogenic agent, the amount, or if appropriate amounts, thereofbeing effective to cause proliferation of cardiomyocytes within thesubject's heart so as to thereby treat the disorder.
 47. The method ofclaim 43, wherein the composition is administered intramyocardially orintracoronarily.
 48. The method of claim 43, wherein the composition isadministered via a stent, a scaffold, or a slow-release formulation. 49.A method of treating a subject suffering from a disorder of a tissueinvolving loss and/or apoptosis of cells of the tissue which comprisesadministering to the subject a composition comprising an amount of anagent which induces phosphorylation and/or activation of protein kinaseB, the composition being administered in an amount effective to causeproliferation of the cells and/or inhibit apoptosis of the cells of thetissue within the subject so as to thereby treat the disorder.
 50. Themethod of claim 49, wherein the agent is human stromal derivedfactor-1α, human stromal derived factor-1β, or human stromal derivedfactor-1γ.
 51. The method of claim 49, wherein the tissue is hearttissue and the cells are cardiomyocytes.
 52. The method of claim 51,wherein the disorder from which the subject is suffering comprisesmyocardial infarction, congestive heart failure, chronic ischemia, orischemic disease.
 53. The method of claim 49, wherein the tissue isheart tissue and the cells are progenitors of cardiomyocytes or stemcells that differentiate to cardiomyocytes.
 54. The method of claim 49,wherein the tissue is heart muscle, striated muscle, liver, kidney,neuronal or gastrointestinal tissue.
 55. The method of claim 49, whereinthe agent is insulin, endothelin-1, urocrotin, cardiotropin-1,erythropoietin, leukemia inhibitory factor-1, tumor necrosisfactor-alpha.
 56. The method of claim 49, further comprisingadministering an amount of one or more of a human granulocyte-colonystimulating factor, a human stromal-derived factor-1, a humangranulocyte macrophage-colony stimulating factor, a human interleukin-8,a human vascular endothelial growth factor, a human fibroblast growthfactor, a human Gro family chemokine, human endothelial progenitorcells, or a pro-angiogenic agent, the amount, or if appropriate amounts,effective to cause proliferation of the cells and/or inhibit apoptosisof the cells of the tissue of the subject so as to thereby treat thedisorder.
 57. A composition comprising a human stromal-derived factor-1and a human granulocyte-colony stimulating factor.
 58. The method ofclaim 49, wherein the composition is administered intramyocardially orintracoronarily.
 59. The method of claim 49, wherein the composition isadministered via a stent, a scaffold, a slow-release formulation,intramuscularly, intravenously, intra-arterially, or sub-cutaneously.60. A method of treating a subject suffering from a disorder of a tissueinvolving loss and/or apoptosis of cells of the tissue which comprisesadministering to the subject a composition comprising an amount of anagent which induces phosphorylation and/or activation of anextracellular signal-regulated protein kinase, the composition beingadministered in an amount effective to inhibit apoptosis and/or causeproliferation of the cells of the tissue within the subject so as tothereby treat the disorder.
 61. The method of claim 60, wherein theagent is human stromal derived factor-1α, human stromal derivedfactor-1β, or human stromal derived factor-1γ.
 62. The method of claim60, wherein the tissue is heart tissue and the cells are cardiomyocytes.63. The method of claim 62, wherein the disorder from which the subjectis suffering comprises myocardial infarction, congestive heart failure,chronic ischemia, or ischemic disease.
 64. The method of claim 60,wherein the tissue is heart tissue and the cells are progenitors ofcardiomyocytes or stem cells that differentiate to cardiomyocytes. 65.The method of claim 60, further comprising administering an amount ofone or more of a human granulocyte-colony stimulating factor, a humanstromal-derived factor-1, a human granulocyte, macrophage-colonystimulating factor, a human interleukin-8, a human vascular endothelialgrowth factor, a human fibroblast growth factor, a human Gro familychemokine, human endothelial progenitor cells, an activator of proteinkinase B, or a pro-angiogenic agent, the amount, or if appropriateamounts, thereof being effective to inhibit apoptosis and/or causeproliferation of the cells of the tissue within the subject so as tothereby treat the disorder.
 66. The method of claim 62, wherein theagent is administered intramyocardially or intracoronarily.
 67. Themethod of claim 60, wherein the agent is administered via a stent, ascaffold, or a slow-release formulation, intramuscularly, intravenously,intra-arterially, or sub-cutaneously.
 68. A method of treating a subjectsuffering from a disorder of a tissue involving loss and/or apoptosis ofcells of the tissue which comprises administering to the subject acomposition comprising an amount of an agent which induces activation ofCXCR4, the composition being administered in an amount effective tocause proliferation of the cells and/or inhibit apoptosis of the cellsof the tissue within the subject so as to thereby treat the disorder.69. The method of claim 68, wherein the tissue is heart tissue and thecells are cardiomyocytes.
 70. The method of claim 69, wherein the agentis administered intramyocardially or intracoronarily via a stent, ascaffold, or a slow-release formulation.
 71. The method of claim 68,wherein the agent is administered systemically.